LIBRARY 
CALIFORNIA 


01 


MEDICAL    SCHOOL 


COLLEGE  OF   PHARMACY 


CHEMISTRY 
DEPARTMENT 


California  GoJ'e^®  of  Pharmac 


PRACTICAL   PHYSIOLOGICAL   CHEMISTRY. 


1st  Edition  -     -     1904. 

2nd  Edition  1908. 

3rd  Edition  1913. 

4th  Edition  1914. 

5th  Edition  -     1919. 

(Completely  revised  and  enlarged.) 


Practical   Physiological 
Chemistry. 


SYDNEY  W.  QOLE,  M.A. 

Trinity  College,   Cambridge. 
University  Lecturer  in  Medical  Chemistry,  Cambridge. 


FIFTH   EDITION. 


With  an  INTRODUCTION  by 

F.    G.    HOPKINS,    M.B.,    D.Sc.,    F.R.C.P.,    F.R.S., 

Professor  of  Biochemistry ;  Fellow  and  Praelector 

of  Trinity  College  ;    Hon.  Fellow  of 

Emmanuel  College,  Cambridge. 


California  CoISe^;©  of  Pharmacy 


ST.  Louis  : 
C.    V.    MOSBY    COMPANY 


7? 


TO   THE 

PRESENT   AND    PAST   MEMBERS 

AND   THE 

SECRETARY 

OF   THE 

MEDICAL   RESEARCH    COMMITTEE 

this  book  is  humbly 

DEDICATED 

by  the  Author  as  a  very  slight  token  of  gratitude  for 

generous  help  received  and  in  appreciation  of 

the  brilliant  results  already  achieved 

in  many  fields  due  to 

their  inception  of 

new  methods 

of  attack. 


INTRODUCTION. 

MY  colleague's  book,  in  its  earlier  editions,  received  so  hearty  and 
so  general  a  welcome  that  any  personal  words  of  recommendation 
seem  now  uncalled  for.  In  this  edition,  however,  it  emerges  as 
a  work  of  a  somewhat  different  order. 

Like  all  good  books  which  deal  with  a  progressive  subject,  it 
has  felt  the  growth  impulse,  and,  notwithstanding  the  exceptional 
nature  of  the  times,  material  for  growth  has  during  the  last  few 
years  accumulated  in  abundance.  If  this  edition  has  largely  out- 
grown its  predecessors  the  increase  is  not  only  desirable,  but,  in 
my  opinion,  necessary. 

Progress  in  any  science  calls  continuously  for  new  methods, 
and  for  extension  and  improvement  in  technique.  In  the  growth 
of  any  branch  of  knowledge  there  are,  indeed,  periods  when  the 
development  of  technique  becomes  the  most  pressing  of  needs,  and 
its  success  the  best  measure  of  progress.  Biochemistry  has  been 
successfully  passing  through  such  a  period.  Its  methods  have 
been  greatly  multiplied,  extended  and  improved.  We  are  now 
beginning  to  reap  the  reward  in  the  accumulation  of  accurate 
quantitative  data.  One  need  peculiar  to  biochemistry,  that  of 
following  changes  in  living  tissues  without  terminating  the  life  of 
the  animal,  or  harming  the  human  subject,  has  now  been  largely 
met,  at  least  so  far  as  studies  of  the  blood  are  concerned.  This  is 
due  to  the  success  of  micro-methods  of  analysis.  So  significant  are 
some  of  the  results  which  can  be  obtained  that  we  may  hope  to  see 
the  methods  become  as  general  in  connexion  with  medical  diagnosis 
as  the  use  of  the  stethoscope  or  the  electrocardiogram. 

It  may  be  good  for  the  advanced  student,  possessed  of  leisure, 
to  determine  for  himself  the  exact  conditions  necessary  for  success 
in  the  use  of  a  given  method.  For  two  classes,  however,  it  is  highly 
desirable  that  success  should  be  reached  as  immediately  as  possible  : 
for  the  elementary  student,  in  order  that  his  faith  may  not  be 
weakened,  and  for  the  research  worker  not  specially  versed  in 
chemical  technique,  who,  with  limited  time,  wishes  to  apply  a 
method  to  medical  or  biological  problems.  Each  of  these  will  be 


VI.  INTRODUCTION. 

the  better  for  descriptions  which  secure  against  failure.  Such  are 
the  descriptions  found  in  this  book.  Indeed,  the  chief  satisfaction 
I  derive  from  being  allowed  to  write  this  foreword  arises  from  the 
opportunity  it  gives  me  of  bearing  witness  to  the  fact  that  the 
author  has  always  a  first-hand  acquaintance  with  his  subject  matter. 
In  connexion  with  the  newer,  and  less  familiar,  tests  and  methods 
the  directions  are  not  copied  from  elsewhere,  not  even  (when  they 
are  due  to  others)  from  the  descriptions  of  their  originators.  They 
have  been  written  at  the  laboratory  bench,  step  by  step  with  the 
successful  accomplishment  of  the  process  they  describe.  When 
success  has  seemed  doubtful,  or  too  difficult  of  attainment,  the 
method  has  found  no  place  in  the  book.  Older  methods  have  often 
been  modified  in  detail,  as  a  result  of  long  experience  of  their  use  in 
practical  classes.  On  the  other  hand  not  a  few  of  the  processes 
described  are  original.  It  is  indeed  a  pleasant  duty  to  emphasise 
the  fact  that  the  book  is  used  as  a  medium  for  the  publication  of  a 
considerable  amount  of  patient  research  work  requiring  abilities  of 
a  special  order.  It  is  to  be  trusted  that  this  work  will  receive  the 
same  degree  of  recognition  that  it  undoubtedly  would  if  it  were 
published  through  the  more  ordinary  channels  of  the  scientific 
periodicals. 

Some  of  the  sections  are  intended  for  purposes  wider  than  that 
of  class  instruction  alone.  In  the  chapter  on  the  preparation  of  the 
amino-acids  for  instance,  Mr.  Cole  has  drawn  on  the  collective 
experience  of  many  workers  in  my  laboratory.  The  descriptions 
are  unique  in  their  wealth  of  detail,  and  I  feel  confident  that  the 
preparation  of  these  compounds  by  the  methods  described  can  be 
undertaken  with  every  prospect  of  success  by  all  workers.  A 
supply  of  pure  amino-acids  is  so  important  for  the  prosecution  of 
many  lines  of  research  that  the  inclusion  of  the  chapter  will  be 
welcomed  by  many  who  have  been  disappointed  at  the  results 
of  their  previous  attempts.  Of  my  own  knowledge  I  can  testify 
to  the  success  that  has  attended  the  preparation  of  histidine  and 
tryptophane,  for  example,  by  junior  laboratory  attendants  following 
the  descriptions  here  published. 

In  my  experience  the  teaching  of  practical  biochemistry  to 
students  of  physiology  presents  a  difficulty  less  felt  in  the  practical 
teaching  of  the  other  branches  of  the  science.  A  successful  histo- 


INTRODUCTION.  Vll. 

logical  preparation,  or  the  neat  accomplishment  of  a  graphic  record, 
yields  immediate  satisfaction  to  a  student  biologically  inclined.  He 
is  dealing  directly  with  the  animal,  and  the  result  seems  an  end  in 
itself.  It  is  otherwise  with  tests  and  estimations  carried  out  as 
mere  exercises.  The  interest  of  a  quantitative  result,  which  may 
be  great  when  it  is  obtained  during  an  actual  study  of  metabolism, 
seems  remote  to  the  student  who  works  without  any  such  stimulus. 
Yet  in  large  chemical  classes  it  is  almost  impossible  to  provide 
closer  touch  with  the  animal,  and  interest  cannot  always  be  secured 
by  maintaining  an  exact  correspondence  in  the  sequence  of  lectures 
and  classes.  It  is  desirable  therefore  that,  even  in  a  book  with  aims 
that  are  avowedly  practical,  there  should  be  some  judicious  reference 
to  theory  and  to  the  actual  significance  of  results.  In  the  present 
work  this  end  seems  to  be  reached  without  undue  consumption  of 
space. 

The  book  in  its  present  form,  while  very  fully  covering  the 
ground  required  by  the  medical  student,  can  be  profitably  used  by 
all  who  seek  for  accurate  and  full  descriptions  of  biochemical 
methods,  whether  for  use  in  medical  diagnosis  or  in  biological 
researches.  The  earlier  editions  of  the  book  have  been  used  by  the 
students  at  the  Agricultural  Laboratory  here,  and  in  its  present 
form  it  would  appear  to  be  highly  suited  to  the  needs  of  those 
engaged  in  the  study  of  animal  nutrition. 


F.   GOWLAND   HOPKINS. 


BIOCHEMICAL    DEPARTMENT, 
CAMBRIDGE. 


PREFACE  TO   THE   FIFTH   EDITION. 


I  much  regret  the  delay  in  publication  which  has  resulted  in 
the  book  being  out  of  print  for  over  a  year.  But  the  pressure  of 
extra  work  during  the  absence  of  most  of  my  colleagues  on  worthier 
occupations  has  prevented  me  devoting  the  time  necessary  for  the 
complete  revision  that  I  desired.  A  good  many  of  the  methods 
described  in  the  last  edition  were  already  obsolete  by  1917,  and  it 
was  therefore  impossible  to  reprint  the  book  and  still  claim  that 
it  represented  the  most  modern  technique. 

I  have  added  a  considerable  amount  of  new  material,  the  most 
important  being  a  chapter  on  the  properties  of  solutions  in  which 
particular  attention  is  paid  to  the  hydrogen-ion  concentration  and 
to  the  colloidal  state  :  a  chapter  on  the  preparation  and  properties 
of  certain  of  the  amino-acids  :  on  the  preparation  and  hydrolysis 
of  nucleic  acid  :  sections  on  the  asymmetric  carbon  atom  and  on  the 
theory  of  the  polarimeter :  on  the  action  of  intestinal  bacteria  on 
proteins  :  on  autolysis  :  on  the  action  of  oxidase  systems  and  many 
new  quantitative  methods  related  to  enzyme  action  and  blood, 
urinary  and  gastric  analyses.  A  good  deal  of  this  material  has  not 
been  published  previously,  but  the  methods  have  stood  the  test  of 
routine  class  work  at  Cambridge,  and  it  is  trusted  that  they  will 
not  fail  when  tried  elsewhere. 

With  the  exception  of  the  exercises  on  the  preparation  of  the 
amino-acids  and  on  the  hydrolysis  of  nucleic  acid,  the  book  represents 
the  course  in  Practical  Physiological  Chemistry  for  Medical  Students 
at  Cambridge..  It  may  be  objected  that  the  course  is  overburdened 
with  analytical  exercises.  They  are  inserted  for  two  reasons  that 
seem  important  to  my  Chief,  Prof.  F.  G.  Hopkins,  and  to  myself. 
In  the  first  place  they  have  considerable  educational  value  :  one  is 
enabled  to  train  the  student  to  make  accurate  observations  and 
pay  attention  to  the  effect  of  variations  in  conditions  on  results. 


PREFACE.  IX. 

Secondly,  it  is  hoped  that  the  student,  having  acquired  the  technique 
necessary  to  determine  the  course  of  the  metabolic  changes  in  the 
normal  individual,  will  be  encouraged  to  extend  his  observations 
to  those  occurring  in  disease.  Progress  in  medical  science  is  largely 
dependent  on  the  statistical  method.  I  am  convinced  that  a  con- 
siderable body  of  trained  medical  men  making  accurate  analyses 
of  the  abundant  clinical  material  that  must  inevitably  come  their 
way  will  advance  our  knowledge  much  more  rapidly  than  a  few 
isolated  specialists,  who  are  apt  to  confine  their  attention  to  subjects 
in  advanced  disease.  It  is  the  border-land  between  health  and  ill- 
health  that  particularly  requires  exhaustive  investigation,  and 
for  its  exploration  a  whole  army  is  required.  We  can  safely  rely 
on  the  Medical  Research  Committee  for  the  Staff  work  necessary 
for  the  proper  co-ordination  of  the  results. 

I  am  much  indebted  to  my  friends  and  colleagues,  Mr.  H. 
Raistrick  and  the  Hon.  H.  Onslow,  for  valuable  help  in  the  chapter 
on  the  preparation  of  the  amino-acids,  and  for  many  suggestions 
made  after  reading  the  proofs  of  the  early  chapters.  Miss  E.  C. 
Travell  has  given  me  great  assistance  in  the  preparation  of  the 
index. 

To  ensure  a  supply  of  the  necessary  apparatus  and  materials, 
arrangements  have  been  made  by  Messrs.  Baird  and  Tatlock 
(London),  by  which  they  hope  shortly  to  be  able  to  maintain  a  stock 
of  all  the  apparatus  and  chemicals  mentioned.  I  am  indebted  to 
them  for  kindly  supptying  a  considerable  number  of  new  illus- 
trations of  the  apparatus  used  in  the  Laboratory. 

Finally,  I  wish  to  record  my  sincere  gratitude  to  Prof.  Hopkins 
for  the  many  valuable  criticisms  and  suggestions  that  have  helped 
me  to  develop  what  he  is  pleased  to  consider  a  satisfactory  course 
for  the  students  attending  his  lectures. 


SYDNEY  W.   COLE. 


BIOCHEMICAL  LABORATORY, 

CAMBRIDGE, 
May,  1919. 


CONTENTS. 


CHAPTER  I. 

PAGE 

THE  PROPERTIES  OF  SOLUTIONS         i 

A.  Colloids  and  Crystalloids      . .         . .         . .  . .  i 

B.  Diffusion  and  Dialysis           . .          . .          . .  . .  2 

C.  Osmotic  Pressure        . .         . .         . .         . .  . .  3 

D.  Freezing  Point            . .         . .         . .         . .  . .  5 

E.  The  Electrical  Properties  of  Colloids         . .  . .  9 

F.  The  Precipitation  of  Colloids          . .         . .  . .  10 

G.  The  Concentration  of  Hydrogen-Ions        . .  . .  14 
H.    Ampholytes  or  Amphoteric  Electrolytes  . .  . .  30 

CHAPTER  II. 

THE  PROTEINS 33 

A.  Definition         . .         . .         . .         . .         . .  . .  33 

B.  Classification    . .         . .         . .         . .         . .  . .  33 

C.  General  Reactions      . .         . .         . .         . .  . .  35 

D.  Colour  Reactions        . .         . .         . .         . .  . .  38 

E.  The  Heat  Coagulation  of  Albumins  and  Globulins  42 

F.  The  Properties  of  Albumins  and  Globulins  . .  45 

G.  The  Chemistry  of  Egg- White          . .         . .  . .  49 

H.    The  Metaproteins       . .         . .         . .         . .  . .  51 

I.      The  Albumoses  or  Proteoses  and  Peptones  . .  52 

J.     The  Gluco-Proteins    . .         . .         . .         . .  . .  57 

K.    The  Reactions  of  certain  Albuminoids     . .  . .  58 

CHAPTER  III. 

THE   NUCLEOPROTEINS,    NUCLEINS   AND    NUCLEIC   ACIDS  .  .  60 


CONTENTS.  xi, 

CHAPTER  IV. 

PAGE 

THE  PREPARATION  AND  PROPERTIES  OF  CERTAIN  AMINO- 

Acms 67 

CHAPTER  V. 

THE  CARBOHYDRATES    . .         . .         . .         . .         . .         . .  100 

A.  The  Monosaccharides             . .         . .         . .         . .  100 

B.  The  Disaccharides 113 

C.  The  Polysaccharides 117 

D.  The  Quantitative  Estimation  of  the  Carbohydrates  125 

E.  The  Theory  and  Use  of  the  Polarimeter  . .         . .  143 

F.  Optical  Activity  and  the  Asymmetric  Carbon  Atom  147 

CHAPTER  VI. 

THE  FATS,  OILS  AND  LIPINES..         ..         ..         ..         ..  153 

CHAPTER  VII. 

THE  CHEMISTRY  OF  SOME  FOODS        166 

A.     Milk 166 

C.  Cheese 172 

D.  Potatoes           173 

E.  Flour 173 

F.  Bread 174 

G.  Meat  (Muscle) 175 

CHAPTER  VIII.  - 

THE  COMPOSITION   OF  THE   DIGESTIVE   JUICES  AND  THE 

ACTION  OF  CERTAIN  ENZYMES        183 

A.  Saliva 186 

B.  Ptyalin 186 

C.  Gastric  Juice   . .         . .         . .         . .         . .         . .  194 

D.  Pepsin   . .         . '.         201 


Xll.  CONTENTS. 

CHAPTER  viii. — continued. 

PAGE 

E.  Rennin  and  the  Clotting  of  Milk      . .         . .  . .  207 

F.  Trypsin             . .          . .          . .          . .          . .  . .  210 

G.  Erepsin             . .         . .         . .         . .         . .  . .  218 

H.    Amylopsin                    . .         . .         . .         . .  . .  220 

I.      Maltase,  Lactase  and  Sucrase         . .         . .  . .  221 

J.     Bacterial  Decomposition  in  the  Intestine . .  . .  222 

K.    Autolysis          . .         . .         . .         . .         . .  . .  228 

L.     Oxidases.  Peroxidases  and  Tyrosinase        . .  . .  230 


CHAPTER  IX. 

THE  COAGULATION  OF  THE  BLOOD 234 

.  CHAPTER  X. 

THE  RED  BLOOD  CORPUSCLES  AND  THE  BLOOD  PIGMENTS  238 

A.  The  Laking  of  Blood 238 

B.  Haemoglobin  and  its  Derivatives  . .         . .         . .  240 

C.  The    Spectroscopic    Examination    of    the    Blood 

Pigments  . .         . .         . .         . .         . .         . .  243 

D.  Blood  Constituents  and  their  Analysis    . .         . .  249 

CHAPTER  XI. 

THE  CONSTITUENTS  OF  BILE  . .        ,. .         . .         . .         . .  264 

'    CHAPTER  XII. 

URINE  AND  ITS  CHIEF  CONSTITUENTS.  .         . .         . .         . .  270 

A.  The  Average  Composition     . .         . .         . .         . .  270 

B.  The  Physical  Chemistry  of  the  Urine       . .         . .  271 

C.  The  Pigments  of  Urine         278 

D.  The  Inorganic  Constituents  . .         . .         . .         . .  280 

E.  Urea  286 


CONTENTS.  Xlll. 

CHAPTER  xii. —continued. 

PAGE 

F.  Uric  Acid         292 

G.  Purine  Bases,  other  than  Uric  Acid. .         . .         . .  298 

H.    Creatinine  and  Creatine         . .          . .          . .          . .  298 

I.      Ammonia          . .          . .          . .          . .          . .          . .  301 

J.     Hippuric  Acid             . .         . .         . .         . .         . .  301 

K.    Certain  Constituents  of  Abnormal  Urines. .         ..  302 

L.     Urinary  Sediments 319 

CHAPTER  XIII. 

THE  QUANTITATIVE  ANALYSIS  OF  URINE 321 

A.  Total  Nitrogen            321 

B.  Ammonia          . .          . .          . .          . .          . .          . .  329 

C.  Ammonia  and  Amino-Acids . .          ..          ..          ..  333 

D.  Urea 334 

E.  Creatinine  and  Creatine        . .         . .         . .         . .  336 

F.  Uric  Acid         341 

G.  Glucose             . .         . .         . .         . .         . .         . .  344 

H.    The  Acetone  Bodies 347 

I.      Chlorides           352 

J.     Phosphates 354 

K.    Sulphates          355 

L.     Albumin            35$ 

M.    Diastase            . .         . .         . .          .  .          . .          . .  359 

CHAPTER  XIV. 

THE  DETECTION  OF  SUBSTANCES  OF  PHYSIOLOGICAL  INTEREST  361 

A.  Fluids    ..         .,         361 

B.  Solids     ..         .. 37° 

APPENDIX. 

Chart  for  Spectroscopic  Absorption  Bands 372 

Chart  for  Recording  Urinary  Analysis                      . .         . .  374 

Weights   and    Measures 375 


XIV.  CONTENTS. 

APPENDIX — continued. 

PAGE 

Tension  of  Aqueous  Vapour     . .         . .  . .  . .  . .  376 

Atomic    Weights..         ..         ..         ..  ..  ..  ..  376 

Specific  Gravity  Tables             . .         . .  . .  . .  . .  377 

Boiling  Points      . .         . .         . .         . .  . .  . .  . .  379 

Standard  Acids  and  Alkalies   . .         . .  . .  . .  . .  380 

Pipettes  and  Burettes   . .         . .         ...  . .  . .  . .  381 

Fume  Absorber  . .         . .         . .         . .  . .  . .  . .  383 

Colorimeters         . .         . .         . .         . .  . .  . .  . .  384 

Micro-balance       . .         . .         . .         . .  . .  . .  . .  388 

Preparation  of  Reagents            . .         . .  . .  . .  . .  389 

Index         . .         . .         . .         . .         . .  . .  . .  . .  392 

Logarithm  Tables           . .         . .         . .  . .  . .  Back  cover 


ILLUSTRATIONS    AND    FIGURES. 


FIG.  PAGE 

1.  Beckmann's  freezing  point  apparatus  . .  . .  . .  8 

2.  Beckmann's  thermometer            . .         . .  . .  . .  8 

3.  Arrangement  of  tubes  in  Comparator    . .  . .  . .  20 

4.  Cole  and  Onslow's  Comparator  . .         . .  . .  . .  21 

5.  Dreyer's  dropping  pipette            . .         . .  . .  . .  23 

6.  Paraffined  bottle  for  storing  standard  alkali    . .  . .  25 

7.  Reflux  condenser  . .         . .         . .         . .  . .  . .  72 

8.  Distillation  in  vacuo          . .         . .         . .  . .  . .  73 

9.  Connexions  of  vacuum  pump      . .         . .  . .  . .  74 

10.  Saturation  with  dry  hydrochloric  acid  gas  . .  . .  75 

11.  Buchner  funnel  and  filtering  flask          . .  . .  . .  76 

12.  Removal  of  hydrogen  sulphide  by  air  current. .  . .  90 

13.  Distillation  in  vacuo  with  Claisen  flask ..  ..  ..  99 

I3a.  Apparatus  for  Benedict's  method           . .  . .  . .  128 

14.  Burette  with  accessory  tip          . .         . .  . .  . .  130 

15.  Apparatus  for  maintaining  a  standard  heating  power  136 

16.  Filtering  apparatus  for  reduced  copper. .  . .  . .  137 

17.  Curve  of  copper  values  for  glucose       . .  . .  . .  138 

18.  Curve  of  copper  values  for  lactose       . .  . .  . .  140 

19.  Crystal  of  calc  spar           . .         . .         . .  . .  . .  143 

20.  Refraction  in  a  Nicol's  prism      . .         . .  . .  . .  144 

21.  Arrangement  of  a  simple  polarimeter     . .  . .  . .  144 

22.  Plan  of  a  three-field  polarimeter            . .  . .  . .  145 

23.  Appearance  of  field  of  polarimeter          . .  . .  . .  145 

24.  Model  of  carbon  atom      . .         . .         . .  . .  . .  148 

25.  Two  superposable  models            . .          . .  . .  . .  149 

26.  Model  of  an  asymmetric  carbon  atom     . .  . .  . .  149 

27.  Plane  of  symmetry  of  model       . .          . .  . .  . .  150 

28.  Meig's  method  of  fat  extraction  . .         . .  . .  . .  170 

29.  Automatic  measuring  apparatus ..         ..  ..  ..  191 


XVI.  ILLUSTRATIONS  AND   FIGURES. 

FIG.  PAGE 

30.  Zeiss'  direct-vision  spectroscope             . .  . .  . .  243 

31.  Flask  fitted  for  sugar  estimation             . .  . .  . .  254 

32.  Titration  in  atmosphere  of  CO2              . .  . .  . .  255 

33.  Gooch  crucible  and  apparatus               . .  . .  . .  259 

34.  Apparatus  for  Micro- Kjeldahl                . .  . .  . .  261 

35.  Titration  in  CO2-  free  atmosphere          . .  . .  . .  261 

36.  Urinometer              . .         . .         . .         . .  . .  . .  272 

37.  Comparator  for  large   tubes        . .          . .  . .  . .  276 

38.  Gauge  for  calibrating  tubes       . .          . .  . .  . .  276 

39.  Hall's   two-way  tube  for  burettes         . .  . .  . .  322 

40.  Kjeldahl  apparatus :   direct  boiling       . .  . .  . .  325 

.41.  Kjeldahl  apparatus :  steam  distillation  . .  . .  326 

42.  Kjeldahl  apparatus :  alcohol  distillation  . .  . .  328 

43.  Apparatus  for  ammonia  estimation  (Folin)  . .  . .  330 
.44.  Apparatus  for  ammonia  and  urea  (Van  Slyke)  . .  331 

45.  Flask  for  ammonia  and  urea    . .          . .  . .  . .  332 

45 a.  Filtering  flask  and  pump  connexion      . .  . .  . .  343 

46.  Apparatus   for   estimation   of   acetone  (Scott-Wilson)  350 

47.  Esbach's  albuminometer             . .         . .  . .  . .  358 

48.  Ostwald  pipette                . .  381 

49.  Method  of  reading  burette         . .         . .  .  .  . .  382 

50.  Apparatus  for  reading  burette             . .  . .  .  .  382 

51.  Folin's  fume-absorber                 . .         . .  . .  .  .  383 

52.  Duboscq  Colorimeter                    . .         .  .  .  .  . .  384 

53.  Path  of  rays  in  Duboscq  Colorimeter      . .  . .  . .  385 

54.  Kober's  Colorimeter        . .         . .         . .  . .  . .  386 

55.  Torsion  balance     . .         . .         . .         . .  . .  . .  388 


CHAPTER   I. 
THE   PROPERTIES   OF  SOLUTIONS. 

A.    Colloids  and  Crystalloids. 

The  condition  of  a  substance  in  "  solution  "  is  one  that 
differs  considerably  with  different  substances  ;  moreover,  it 
may  differ  with  the  same  substance,  depending  on  the 
method  of  preparing  the  solution.  All  "solutions"  can 
be  regarded  as  suspensions  of  particles  in  the  "solvent." 
The  size  and  nature  of  the  particles  cause  variations  in 
the  physical  properties  of  the  solution. 

There  are  four  classes  of  so-called  "solutions,"  which  are 
not,  however,  very  sharply  differentiated  from  one  another. 
They  are 

(ELECTROLYTES. 
\NON-ELECTROL  YTES. 


COLLOIDS 

(SUSPENSOIDS. 

Probably  the  essential  difference  between  colloids  and 
crystalloids  is  the  size  of  the  particles  suspended  in  the 
fluid.  In  the  crystalloids  these  particles  are  small,  con- 
sisting of  ions  or  single,  relatively  small  molecules.  In  the 
colloids  the  particles  are  large,  either  because  the  molecules 
themselves  are  large,  or  because  they  tend  to  aggregate 
and  form  relatively  large  masses.  The  difference 
between  emulsoids  and  suspensoids  is  probably  that 
suspensoids  are  two  phase  liquids  in  which  the  "  solvent  " 
(external  phase)  does  not  combine  with  the  "  solute  " 
(internal  phase),  that  is  to  say,  the  solute  is  in  real 
suspension  in  the  so-called  solvent.  In  emulsoids,  on  the 


2  THE  PROPERTIES   OF  SOLUTIONS.  [CH.   I. 

other  hand,  we  have  two-phase  liquids,  each  phase  con- 
taining both  components  in  different  concentrations.  The 
solute  is  able  to  combine  to  a  certain  extent  with  the  solvent. 
They  are  intermediate  between  suspensions  and  true  solu- 
tions. In  certain  cases  there  is  evidence  to  show  that  the 
solute  may  be  partially  ionised.  Solutions  of  native  pro- 
teins, starch,  dextrins,  etc.,  are  emulsoids,  whereas  the 
denaturised  proteins  behave  more  like  suspensoids. 

Sorensen's  view  as  to  the  main  differences  between  the  two  types  of 
colloids  is  as  follows  : — 

"  The  suspensoids  show  a  viscosity  differing  but  little  from  that  of  the 
pure  external  phase.  There  is  generally  a  well-marked  difference  in  electrical 
charge  between  the  two  phases.  Only  comparatively  small  concentrations 
of  electrolytes  are  required  to  bring  about  coagulation,  which  is  in  most  cases 
irreversible. 

"The  emulsoids  show  a  great  viscosity  and  power  of  foam  formation; 
the  system  commonly  does  not  show  any  marked  difference  in  electrical  charge 
between  the  two  phases.  Great  concentrations  of  electrolytes  are  commonly 
necessary  to  bring  about  coagulation,  which  is  in  most  cases  reversible." 

B.    Diffusion  and  Dialysis. 

The  difference  between  crystalloids  and  colloids  that 
has  been  most  emphasised  is  the  disparity  in  the  rate  of 
diffusion  of  the  two  substances.  If  a  solution  of  sodium 
chloride  or  glucose  be  separated  from  distilled  water  by 
means  of  a  film  of  collodion,  parchment  paper,  or  gold 
beater's  skin,  the  dissolved  substance  is  found  to  pass 
through  the  membrane,  the  process  being  known  as 
diffusion.  If  a  colloidal  solution  be  tested  in  the  same  way, 
it  will  be  found  to  pass  through  either  very  slowly  or  not  at 
all.  In  other  words,  the  colloids  are  relatively  indiffusible. 
This  is  sometimes  employed  as  a  convenient  method  of 
separating  crystalloids  from  colloids,  and  is  known  as 
dialysis.  It  should  be  noted,  however,  that  abrupt 
transitions  are  not  common  in  nature,  and  that  all  emul- 
soids do  diffuse  through  such  membranes,  though  extremely 
slowly  as  compared  to  crystalloids. 

i.  Preparation  of  collodion  sacs  for  dialysis.  A  convenient 
size  is  made  by  use  of  a  large  boiling  tube  (200  x  15  mm.).  Into  a 
clean,  dry  tube  pour  about  10  cc.  of  the  collodion  solution  described 


CH.    I.]  DIALYSIS.  3 

below.  Pour  this  back  into  the  stock,  revolving  the  tube  with  the 
mouth  downwards  so  that  an  even  film  is  left  adherent  to  the  walls 
of  the  tube.  Add  another  portion  of  collodion  solution  and  repeat 
as  before.  Allow  the  film  to  dry,  so  that  it  does  not  stick  to  the 
finger.  When  this  point  is  reached  fill  the  tube  with  cold  water. 
Cut  round  the  rim  of  the  tube  with  a  knife,  pour  off  the  water,  and 
carefully  detach  the  membrane  from  the  side  of  the  tube.  Allow 
water  to  run  between  the  sac  and  the  glass.  By  means  of  a  glass 
rod  with  a  spatulate  end,  and  by  traction  and  twisting,  the  sac  can 
usually  be  removed  from  the  glass  tube.  Fill  the  sac  with  water.  It 
should  be  perfectly  transparent.  A  cork,  bored  with  a  large  hole, 
can  be  tied  into  the  upper  end,  and  by  means  of  this  it  can  be  sus- 
pended in  a  jar  of  distilled  water.  The  secret  of  success  is  to  fill  the 
tube  with  water  at  a  particular  moment,  determined  by  trial  on  each 
specimen  of  collodion.  If  the  water  be  added  too  soon  the  sac  is 
opaque  and  feeble.  If  it  be  added  too  late,  it  is  somewhat  difficult 
to  remove  the  film  from  the  glass  without  damage.  Sacs  prepared 
in  this  way  are  very  much  better  than  those  made  of  parchment 
paper.  They  should  be  kept  wet,  as  on  drying  they  become  porous. 

NOTE. — Preparation  of  Collodion  Solution.  To  3  gm.  of  gun  cotton 
(pyroxylin)  add  75  cc.  of  ether  and  allow  to  stand  for  10  or  15  minutes  in  a  flask 
closed  with  a  cork.  25  cc.  of  ethyl  alcohol  are  then  added,  and  the  pyroxylin 
dissolves  to  a  mobile  fluid,  which  does  not  require  nitration.  It  should  be 
allowed  to  stand  until  all  bubbles  have  disappeared. 

2.  Dialysis.  Mix  2  per  cent,  starch  paste  (see  Ex.  135)  with 
about  one-tenth  of  its  volume  of  saturated  ammonium  sulphate 
solution.  Place  the  mixture  in  a  collodion  sac  and  suspend  this  in 
water  contained  in  a  tall  jar.  Care  must  be  taken  to  avoid  spilling 
any  of  the  mixture  into  the  jar.  Examine  the  "  dialysate  "  (the 
fluid  in  the  jar)  for  starch  by  the  iodine  test  (Ex.  136)  and  for  am- 
monium sulphate  by  means  of  barium  chloride  at  the  end  of  half  an 
hour.  The  starch  test  will  probably  be  negative,  whilst  the  sulphate 
test  will  be  positive.  The  dialysate  should  also  be  examined  for 
starch  after  2  to  7  days. 

C.    Osmotic  Pressure. 

Certain  membranes  can  be  prepared  which  allow  of 
the  passage  of  water  molecules,  but  do  not  allow  dissolved 
substances  to  pass  through  them.  Such  membranes  are 


4  THE    PROPERTIES   OF   SOLUTIONS.  [CH.    I. 

called  "  semi-permeable."  If  a  dissolved  substance,  like 
glucose,  be  separated  from  water  by  means  of  a  semi- 
permeable  membrane,  the  sugar  solution  is  diluted  by 
water  passing  through  the  membrane.  This  process,  the 
passage  of  water  through  a  membrane  into  a  solution,  is 
known  as  <(  osmosis,"  and  is  to  be  carefully  distinguished 
from  "  diffusion,"  the  passage  of  a  dissolved  substance 
through  a  membrane.  If  the  sugar  solution  be  contained  in 
a  vessel  connected  to  a  manometer,  and  arrangements  are 
made  to  keep  the  volume  of  the  solution  constant,  it  is 
found  that  the  water  passing  into  the  vessel  causes  a  rise  of 
pressure.  The  final  pressure  reached  is  known  as  "  the 
osmotic  pressure  "  of  the  solution. 

It  is  not  necessary  here  to  enter  into  the  theories  of 
osmotic  pressure,  but  it  is  important  to  note  that  the 
osmotic  pressure  of  a  solution  depends  on  the  number  of 
particles  in  a  given  volume,  no  matter  whether  these 
particles  be  ions,  molecules,  or  aggregates  of  molecules. 
Thus  the  osmotic  pressure  of  a  dilute  solution  of  sodium 
chloride  is  nearly  double  that  of  an  equimolecular  solution 
of  glucose,  because  in  dilute  solution  the  sodium  chloride  is 
almost  completely  dissociated  into  its  constituent  ions. 
From  these  considerations  it  follows  that  the  osmotic 
pressure  of  a  colloidal  solution  is  extremely  low  in  com- 
parison with  that  of  a  crystalloid  of  the  same  percentage, 
for  the  number  of  particles  in  a  given  volume  of  the  colloidal 
solution  is  very  small  compared  with  that  in  the  crystalloid 
solution. 

For  non-electrolytes  it  has  been  shewn  by  Van  't  Hoff  that  Boyle's  law 
for  gases  can  be  applied  to  solutions,  if  we  substitute  osmotic  pressure  for  gas 
pressure.  It  has  also  been  found  that  the  Law  of  Charles  is  obeyed,  namely, 
that  at  constant  volume  the  pressure  varies  as  the  absolute  temperature. 

It  follows  that  in  dilute  solute  solution 

V.P.  =  R.T. ;       where      V  =  Volume ;          P  =  Osmotic   Pressure ; 
T  =  Temperature  (absolute),      and      R  =  a  Constant. 

Moreover,  it  has  been  shewn  by  Van  't  Hoff  that  R  in  the  case  of  osmotic 
pressure  has  the  same  value  as  in  the  case  of  gases,  that  is,  the  solution  exerts 
the  same  osmotic  pressure  as  the  pressure  that  the  dissolved  substance  would 
exert  if  it  were  gasified  at  the  same  temperature  and  confined  in  the  same 
volume  as  that  of  the  solution.  It  follows  that  one  gramme-molecule  of  a 
non-electrolyte  will  exert  an  osmotic  pressure  at  o°  C.  of  760  mm.  of  mercury 
when  the  volume  of  the  solution  is  22-4  litres. 


CH.    I.]  OSMOTIC    PRESSURE.  5 

If  w  grams  of  a  substance  be  dissolved  in  V  cc.  of  solvent  at  t°  C.  and 
the  osmotic  pressure  produced  be  P.  mm.  of  mercury,  the  volume  at  o°  C.  and 
at  760  mm.  mercury  will  be 

V  x  273  x  P 
(273T~t)  x  760  =  V° 

Now  V0  contains  w  grams  of  substance,  so  22400  cc.  contains 
22400  x  w 


Since  this  weight  of  substance  produces  a  pressure  of  760  mm.  at  o°  C.  when 
in  a  volume  of  22-4  litres  it  follows  that  it  is  the  molecular  weight  of  the 
substance. 

Instead  of  the  formula  V.P.  =  R.T.,  we  must  use  the  following  for  electro- 
lytes, 

V.P.  =  2  *  +  (I0°  "  *)  R.T.,  where  i  is  the  percentage  of  the  substance 
100 

ionised. 

3.  Chemical  garden.  To  a  dilute  aqueous  solution  of  potassium 
ferrocyanide  add  a  particle  of  solid  ferric  chloride.     A  film  of 
Prussian  blue  is  formed  round  the  solid.     This  membrane  is  semi- 
permeable,  and  allows  water  to  pass  in  to  dissolve  the  ferric  chloride. 
The  osmotic  pressure  of  this  solution  being  greater  than  that  of  the 
solution  outside,  water  passes  into  the  cell,  which  expands,  and  may 
assume  remarkable  forms. 

4.  A  drop  of  a  fairly  strong  solution  of  potassium  ferrocyanide 
is  added  to  a  dilute  solution  of  copper  sulphate.     A  semipermeable 
cell  of  copper  ferrocyanide  is  thus  formed  around  the  drop.    The 
osmotic  pressure  of  the  potassium  ferrocyanide  being  greater  than 
that  of  the  copper  sulphate,  pure  water  passes  from  the  sulphate 
into  the  cell.     This  results  in  a  concentration  of  copper  sulphate 
immediately  around  the  cell,  and  blue  striae  can  be  seen  descending 
owing  to  the  greater  density  of  the  strong  copper  sulphate  solution 
thus  formed. 

D.    Freezing  Point. 

The  freezing  point  of  a  solution  of  a  substance  is 
always  lower  than  that  of  the  solvent.  The  depression  of 
the  freezing  point  (A)  depends  on  the  number  of  particles 
in  a  given  volume  of  the  solution.  We  have  already  seen 
that  the  osmotic  pressure  of  a  solution  also  varies  with 
the  number  of  particles  in  a  given  volume  of  the  solution. 
It  therefore  follows  that  A  varies  with  the  osmotic  pressure. 


6  THE   PROPERTIES   OF   SOLUTIONS.  [CH.    I. 

Consequently  the  osmotic  pressure  of  a  solution  is  most  con- 
veniently estimated  by  a  determination  of  its  freezing 
point. 

The  depression  of  the  freezing  point  (A)  for  a  given 
concentration  of  a  substance  varies  with  the  solvent 
employed,  the  relationship  for  non-electrolytes  being 

-  x  M  =  C,  where  w  is  the  weight  of  a  substance  of  mole- 
w 

cular  weight  M  dissolved  in  100  grams  of  the  solvent  and 
C  is  the  "coefficient  of  depression"  for  the  particular 
solvent.  If  5  grms.  of  solvent  are  taken  instead  of  100,  it 
follows  that  since  A  is  proportional  to  the  concentration 

s     x  -  x  M  =  C 


IOO          W 

The  value  of  C  for  water  is  18-6°  C.  :   for  acetic  acid  it  is 
39°  C. 

Van  't  Hoff  ha^s  shewn  that  the  value   of  C  can  be 

2  T2 

calculated  from  the  formula  C  =  -  -  .  where  T  is  the 

loo  L 

absolute  temperature  of  the  freezing  point,  and  L  is  the 
latent  heat  of  fusion  of  the  solvent. 

Thus  for  water  T  =  273 
L  =    80 


° 


So     C  =  2  X  =  18-6°. 

IOO  X  80 

With  non-electrolytes,  therefore,  the  gramme-molecule 
in  1,000  gm.  of  water  causes  a  depression  (A)  of  1-86°  C. 

So  that  --  =  molecular  concentration. 
1-86 

For    electrolytes  :   =  concentrations    of    (ions  + 

i  '86 

molecules),  so  that  if  a  substance  be  ionised  to  the  extent 
of  i  per  cent,  the  molecular  concentration  is 

A  x  loo 
1-86  x  (21  +  loo-  i)  ' 


CH.    I.]  FREEZING   POINT.  7 

The  quantitative  relationship  between  osmotic  pres- 
sure and  A  for  aqueous  solutions  can  be  readily  calculated 
as  follows. 

The  gramme-molecule  in  22-4  litres  gives  an  osmotic 
pressure  of  760  mm.  of  mercury  at  o°  C. 

The  gramme-molecule  in  i  litre  gives  a  A  of  i  -86°  C. 

So  the  gramme-molecule  in  22-4  litres  gives   a   A   of 

1-86  (0  ^ 

=  0-083    C. 

22-4 

So  a  A  of  0-083  C.  corresponds  to  an  osmotic  pressure 
of  760  mm. 

So  a  A  of  o-oo i  C.  corresponds  to  an  osmotic  pressure 
of  9-1  mm. 

Thus  a  5  per  cent,  solution  of  glucose  (Mol.wt.  =  180) 

has  a  A  of  1-86  x  -^-  =  0-517°  C., 

1 80 

and  an  osmotic  pressure  of  51-7  x  9-1  =  470  mm.  Hg. 

The  A  of  Blood  is  about  0-55°  C.,  corresponding  to  an 
osmotic  pressure  of  about  5oomm.Hg. 

Owing  to  the  relatively  small  number  of  particles  in  a 
given  volume  of  a  colloidal  solution,  it  follows  that  the 
freezing  point  of  such  solutions  is  only  very  slightly  lower 
than  that  of  distilled  water.  Since  it  is  very  difficult  to 
remove  the  last  traces  of  electrolytes  by  dialysis,  it  is  not 
easy  to  obtain  reliable  figures  for  the  osmotic  pressure  of 
the  colloids.  Sorensen  has  recently  investigated  the  problem 
and  has  been  successful  in  overcoming  the  technical  diffi- 
culties. He  states  that  the  osmotic  pressure  of  crystalline 
egg-albumin  indicates  a  molecular  weight  of  34,000. 

5.  The  determination  of  the  freezing  point  by  Beckmann's 
method.  (Cryoscopy.) 

Take  the  freezing  point  of  (a)  distilled  water;  (b)  M/5  NaCl 
(1-16  per  cent.) ;  (c)  M/5  glucose  (3-6  per  cent.). 


8 


THE    PROPERTIES    OF    SOLUTIONS. 


[CH.    I. 


Use  the  apparatus  shown  in  fig.  i.  In  the  outer  chamber  (c) 
place  a  mixture  of  ice  and  water  and  solid  sodium  chloride,  or  a 
saturated  solution  of  salt. 


Fig.  i.     Beckmann's  freez- 
ing point  apparatus. 


Fig.  2. 
Beckmann's 

Ther- 
mometer. 


In  the  tube  A  place  enough  distilled  water  to  cover  the  bulb 
of  the  Beckmann  thermometer  D.  This  is  graduated  to  i/iooth°  C. 
and  can  be  read  by  means  of  a  magnifying  glass  to  1/1000°  C.  The 
thermometer  must  not  touch  the  sides  or  bottom  of  the  tube  A. 


CH.    I.]  COLLOIDS.  9 

The  tube  B  serves  as  an  air  jacket  to  A.  Stir  the  water  regularly 
by  means  of  the  (platinum)  stirrer  E.  The  temperature  falls,  and 
then  after  a  time  rises  sharply,  and  remains  steady  for  a  considerable 
time.  The  temperature  to  be  read  is  the  highest  obtained  at  this 
rise.  This  is  the  freezing  point  (W)  of  distilled  water. 

Now  replace  the  water  by  the  fluid,  rinsing  the  tube  out  with 
it  once  or  twice.  Repeat  the  experiment  and  note  the  freezing 
point  (F)  as  before.  W  —  F  =  A. 

NOTES. — i.  It  is  of  the  utmost  importance  to  take  care  to  prevent  too 
great  a  super-cooling  of  the  fluid.  This  should  never  exceed  i  °  C.  If  it  has 
exceeded  this  in  a  preliminary  experiment,  it  must  be  repeated,  and  when  the 
temperature  has  fallen  0-5°  C.  below  the  freezing  point,  a  minute  crystal  of  ice 
must  be  introduced  through  the  side  tube.  These  crystals  are  best  prepared 
by  taking,  in  a  dry  test-tube,  some  hollow  glass  beads  (that  have  been  care- 
fully dried),  adding  a  small  amount  of  the  fluid,  pouring  off  the  excess,  and 
immersing  the  tube  in  a  freezing  mixture.  They  should  be  introduced  by 
means  of  a  pair  of  cooled  forceps. 

2  The  observed  A  is  usually  too  great,  owing  to  the  super-cooling.  The 
simplest  correction  is 

A  corrected  =  A  observed  x   I  i  -  ^-  1 

where  C  =  the  super-cooling  in  degrees. 

3.  To  set  the  thermometer.     Turn  the  thermometer  upside  down,  and  by 
gentle  shaking  mix  the  mercury  in  the  upper  portion  with  that  in  the  capillary 
tube.     Then  place  the  thermometer  in  water  at  about  2°  C.     Give  a  slight 
knock,  and  thus  break  the  mercury  column.     It  is  now  ready  for  use. 

4.  When  reading  the  thermometer  during  an  experiment  it  should  be 
tapped  with  a  piece  of  indiarubber  tubing. 

E.    The  electrical  properties  of  colloids. 

Under  certain  conditions  it  is  found  that  colloidal 
particles  carry  an  electric  charge.  In  some  cases  they 
exhibit  electrical  conductivity,  due  to  the  fact  that  the 
substances  are  partially  ionised.  But  even  if  they  do  not 
exhibit  this  phenomenon  it  is  often  found  that  they  tend  to 
move  towards  one  of  the  poles  when  a  strong  ( 100  volts)  con- 
stant current  is  sent  through  the  solution.  In  some  cases 
this  movement  ("  kataphoresis ")  is  towards  the  anode,  i.e.* 
the  particles  carry  a  negative  charge  ;  in  other  cases  it  is 
towards  the  kathode.  It  is  important  to  note  that  the 
direction  of  the  migration  can  be  changed  in  many  cases  by 
varying  the  reaction  of  the  fluid  in  which  the  colloid  is 
suspended.  Thus  metaproteins,  albumins,  etc.,  carry  a 


10  THE    PROPERTIES    OF   SOLUTIONS.  [CH.    I. 

positive  charge  in  acid  solution  and  a  negative  charge  in 
alkaline  solution.  At  some  particular  reaction  they  seem 
to  be  electrically  neutral,  i.e.  kataphoresis  cannot  be 
observed.  This  reaction  is  known  as  "  the  iso-electric 
point  "  of  the  particular  colloid.  It  is  discussed  in  more 
detail  on  p.  31. 

F.    The  precipitation  of  colloids. 

Colloids,  as  we  have  seen,  are  two  phase  solutions. 
One,  the  solid,  phase  contains  a  high  concentration  of  the 
solute  and  a  low  concentration  of  the  solvent  :  the  other, 
the  liquid,  phase  contains  a  low  concentration  of  the 
solute  in  the  solvent.  By  certain  changes  in  the  condi- 
tions the  solid  phase  can  be  dehydrated,  so  that  the  solution 
may  become  opalescent.  An  increase  of  this  effect  may 
result  in  the  formation  of  particles  visible  to  the  naked 
eye,  or  even  a  dense  precipitate  that  can,  in  some  cases, 
be  removed  completely  by  filtration.  In  some  cases  this 
precipitate  can  be  "  dissolved  "  or  "  dispersed  "  by  revert- 
ing to  the  original  conditions.  In  other  cases  the  change  is 
irreversible,  the  material  having  been  "  coagulated." 

It  is  impossible  to  discuss  fully  the  various  conditions 
that  tend  to  cause  aggregation  (i.e.  precipitation)  on  the 
one  hand,  or  dispersion  (i.e.  solution)  on  the  other,  since 
they  vary  considerably  with  different  colloids.  But  it  is 
important  to  note  that  many  cases  can  be  explained  fairly 
satisfactorily  on  the  theory  that  the  dispersed  or  dissolved 
condition  of  a  colloid  is  due  to  the  fact  that  it  carries  an 
electric  charge,  the  removal  of  which  causes  precipitation. 
Some  examples  of  this  are  given  below  : — 

(a)  By  colloids  with  an  opposite  electrical  charge. 

If  ferric  chloride  be  thoroughly  dialysed  a  colloidal 
suspension  of  ferric  hydroxide  is  obtained,  generally  known 
as  "  dialysed  iron."  This  carries  a  positive  charge.  If 
this  be  added  to  certain  albumins  which  carry  a  negative 
charge  the  two  colloids  mutually  precipitate  one  another. 
This  gives  us  a  valuable  method  for  removing  certain 
proteins  from  solution.  (See  Ex.  310.) 


CH.   I.] 


ISO-ELECTRIC   POINT. 


11 


(b)  By  changing  the  reaction  of  the  fluid. 

In  acid  solution  most  colloids  "  adsorb  "  the  positively 
charged  and  readily  diffusible  hydrogen  ions  and  acquire  a 
positive  charge.  In  alkaline  solutions  they  adsorb  hydroxyl 
ions  and  become  negative.  Many  proteins  are  therefore 
soluble  both  in  acids  and  alkalies,  but  at  some  particular 
reaction  of  the  fluid  they  adsorb  equal  numbers  of  H  and 
OH  ions,  lose  their  charge,  and  are  precipitated.  The 
exact  reaction  at  which  this  takes  place  varies  with  different 
colloids,  and  is  the  above-mentioned  iso-electric  point. 
Another  way  of  explaining  this  phenomenon  will  be  found 
on  p.  31. 

6.    The  determination  of  the  iso-electric  point  of  casein. 

Into  a  50  cc.  measuring  flask  place  0-3  gm.  of  pure  casein 
(Hammersten's).  Add  about  25  cc.  of  distilled  water,  previously 
warmed  to  about  40  C.  and  exactly  5  cc.  of  N.  sodium  hydroxide. 
Agitate  till  the  casein  dissolves,  taking  care  to  prevent  frothing. 
Rapidly  add  5  cc.  of  N.  acetic  acid,  mix,  cool,  and  make  up  to  50  cc. 
with  distilled  water.  A  faintly  opalescent  solution  of  casein  in 
o-i  N.  sodium  acetate  is  thus  obtained. 

Make  up  the  following  series  of  tubes,  using  clean  dry  test-tubes. 


Tube  No. 

i 

2 

3 

4 

5 

6 

6 

8 

9 

cc.    Casein   in   o-i    N. 

sod.  acetate 

i 

I 

i 

i 

i 

i 

I 

i 

i 

cc.  Distilled  water 

8-38 

775 

8-75 

8-5 

8 

7 

5 

i 

7'4 

cc.  o-oi  N.  acetic  acid 

0-62 

1-25 

? 

;i 

cc.  o-i  N.  acetic  acid  .  . 

O'25 

o-5 

i 

2 

4 

8 

N.  acetic  acid 

& 

jfr 

1-6 

Place  the  casein  solution  in  the  tubes  first,  then  the  water,  and 
mix.  Now  add  the  acetic  acid  to  the  first  tube  and  shake  immedi- 
ately. Then  add  the  acid  to  the  second  tube  and  shake  this,  and 
so  on.  Examine  the  tubes  at  intervals  and  record  observations  as 
below. 

o  =  no  change.         +  =  opalescence.          x  =  precipitate. 


12 


THE  PROPERTIES  OF  SOLUTIONS. 


[CH.  I. 


Tube  No. 

i 

2 

3 

4 

5 

6 

7 

8 

9 

On  mixing 

0 

0 

+ 

+  + 

+  +  + 

+  + 

+ 

+ 

o 

After  10  mins.     .  . 

0 

0 

+ 

+  +  + 

XXX 

X  X 

+  + 

+ 

o 

After  20  mins.     .  . 

o 

o 

+ 

X 

XXX 

X  X 

+  + 

+ 

0 

The  precipitation  is  greatest  in  tube  5. 

The  concentration  of  hydrogen  ions  (see  p.  19)  can  be  calculated 
approximately  from  the  following  formula,  no  allowance  being  made 
for  the  acidity  of  the  casein. 

.        K  (acetic  acid  in  mols.  per  litre) 
a  (sodium  acetate  mols.  per  litre) 
(H)  =  Hydrogen  ions  in  grams  per  litre. 
K  =  dissociation  constant  of  acetic  acid  =  1-85  x  io~5. 

a  =  dissociation  constant  of  sodium  acetate.  0-87  for  o-oi  N. 

0-79  for  o-i  N. 

The  theoretical  basis  for  the  formula  is  given  on  p.  19. 
Thus  in  tube  5, 

(H)  =  1-85  x  io-5  x  io-2 

_ =  2-13  x  io~5. 

0-87          x  io~2 

Below  are  the  (H)  and  PH  (see  p.  16)  of  the  various  tubes. 


Tube 

(H) 

PH 

Tube 

(H) 

PH 

i 

1-32  x  io~6 

5-88 

6 

4-26  x  io~5 

4'37 

2 

2-66  x  io~6 

575 

7 

8-52  x  io~5 

4-07 

3 

5-32  x  io~6 

5-27 

8 

1-70  x  io~4 

377 

4 

i  -06  x  io~5 

4-97 

9 

3-40  x  io~4 

3'47 

5 

2-13  x  io~5 

4-67 

Still  finer  adjustments  of  the  reaction  can  be  obtained  by  suit- 
ably varying  the  concentration  of  acetic  acid.  The  (H)  can  be 
calculated  from  the  formula. 


CH.    I.] 


COLLOIDS. 


13 


(c)  By  the  addition  of  neutral  salts. 

If  a  suspensoid  carries  a  negative  charge  it  exerts  an 
attraction  for  positively  charged  ions  (kations).  The 
adsorption  of  these  by  the  colloid  may  cause  a  neutralisa- 
tion of  the  charge,  and  therefore  precipitation.  In  such 
cases  it  is  found  that  a  bi-  or  tri-valent  ion  is  very  much 
more  potent  than  a  monovalent  ion.  Thus,  if  a  colloid  is 
negatively  charged  it  may  be  readily  precipitated  by 
BaCLj ;  if  it  carries  a  positive  charge  it  may  be  readily 
precipitated  by  Na2SO4.  The  precipitation  of  an  emulsoid  by 
a  large  excess  of  neutral  salt,  such  as  by  saturation  with 
ammonium  sulphate,  is  probably  a  different  phenomenon. 

7.    The  precipitating  effect  of  various  ions  on  colloids. 

Prepare  a  solution  of  casein  in  o-i  N.  sodium  acetate  as  de- 
scribed in  Ex.  6. 

To  2  cc.  add  17-5  cc.  of  distilled  water,  and  then  0-5  cc.  of 
0*1  N.  acetic  acid  and  mix  quickly.  A  solution  of  casein  is  thus 
obtained,  alkaline  to  the  isoelectric  point,  and  therefore  carrying  a 
negative  charge.  Divide  the  solution  into  four  equal  parts  and 
place  them  into  four  clean  tubes  labelled  -i,  -2,  -3  and  -4.  To 
another  2  cc.  of  the  original  solution  of  casein  add  10  cc.  of  distilled 
water  and  8  cc.  of  o-i  N.  acetic  acid,  and  mix  quickly.  An  acid 
solution  of  casein  is  thus  obtained.  Divide  into  four  parts  and 
place  them  into  four  clean  tubes  labelled  -fi,  +2,  +3  and  +4. 

To  the  tubes  marked  i  add  3  drops  of  N.  KC1  (7-45  per  cent.). 

To  the  tubes  marked  2  add  i  drop  of  N.  BaQ2  (10-4  per  cent.). 

To  the  tubes  marked  3  add  i  drop  of  N.  K2SO4  (8*7  per  cent.). 

Mix  the  contents  of  each  tube  and  place  the  set  of  8  tubes  in  a 
water  bath  at  about  35  C.  Examine  them  after  15  minutes,  record- 
ing the  results  as  in  the  previous  exercise. 


X 

o  or  + 
o 


X  X 

o 


14  THE    PROPERTIES    OF   SOLUTIONS.  [cH.    I. 

It  will  be  noted  that  the  electro-negative  colloid  is  most  readily 
precipitated  by  BaCl2,  which  contains  a  di-valent  positive  ion.  The 
electro-positive  colloid  is  most  readily  precipitated  by  K2SO4,  which 
contains  a  di-valent  negative  ion. 

The  tubes  may  now  be  warmed  to  60°  C.,  and  the  further  effect 
noted. 

(d)  By  the  addition  of  compounds  ivith  complex  ions. 

It  is  found  that  colloids  that  carry  a  positive  charge  are 
often  readily  precipitated  by  compounds  with  a  complex 
negative  ion.  Thus,  proteins  in  acid  solution  generally 
carry  a  positive  charge,  and  they  are  precipitated  by 
phosphotungstic,  phosphomolybdic,  tannic,  or  ferrocyanic 
acids.  Probably  the  complex  ions  are  more  readily 
adsorbed  by  the  positively  charged  colloid  than  are  the 
simple  ions.  The  charge  of  the  colloid  thus  being  neutra- 
lised, precipitation  of  the  complex  takes  place. 

If  the  colloid  carries  a  negative  charge  it  is  often  readily 
precipitated  by  compounds  with  a  complex  positive  ion, 
such  as  the  hydrochlorides  of  the  alkaloids,  aromatic 
bases,  etc. 

G.    The  Concentration  of  Hydrogen  ions. 

The  only  satisfactory  method  of  expressing  the  "re- 
action" of  a  fluid  is  in  terms  of  the  concentration  of  hydro- 
gen ion%  per  litre  of  the  fluid.  This  concentration  is  so 
important  as  a  factor  in  the  physiological  properties  of 
fluids  that  the  theory  of  the  matter  should  be  grasped  by 
students  at  an  early  stage  of  their  physiological  studies. 

Pure  distilled  water  is  very  slightly  ionised  into  hydro- 
gen ions  or  hydrions  and  hydroxyl  ions  or  hydroxidions. 

H2O  ~ >  H  +  OH. 

This  dissociation  proceeds  to  an  equilibrium,  in  which, 
according  to  the  laws  of  mass  action, 

(H)  x  (OH) 
(H20)     2- 


CH.  I.]         HYDROGEN  ION  CONCENTRATION  15 

So  (H)  x  (OH)  =  a  constant  x  (H2O). 

The  brackets  indicate  the  concentration  per  litre  of 
ions  or  moles  respectively. 

Since  the  mass  of  undissociated  water  is  enormously 
large  compared  to  the  mass  of  the  free  ions,  it  can  be 
regarded  as  a  constant,  so 

(H)  x  (OH)  =  a  constant. 

This  constant  varies  considerably  with  the  tempera- 
ture. 

At  21°  C.  it  is  or  io~14. 

1 00,000,000,000,000 

Since  hydrions  and  hydroxidions  are  equal  in  number, 

each  has  a  concentration  of or  io~7  per  litre. 

10,000,000 

If  an  acid  be  added  to  distilled  water  the  acid  is  partially 
or  completely  dissociated  into  hydrogen  ions,  and  the 
negative  ions  characteristic  of  the  acid  employed.  In 
such  a  mixture  the  concentration  of  hydrogen  ions  per  litre 
at  21°  C.  is  greater  than  io~7,  and  the  solution  is  "  acid." 
If  (H)  be  increased  to  io~4  it  follows  that  the  concentration 
of  hydroxyl  ions  per  litre  must  be  decreased  to  io~10.  For 
(H)  x  (OH)  =  io-14.  If  an  alkali  be  added  to  distilled 
water  the  base  is  dissociated  into  hydroxyl  ions  and  certain 
positive  ions.  The  concentration  of  hydrogen  ions  per 
litre  at  21  °  C.  is  consequently  less  than  io~7,  and  the 
solution  is  "  alkaline." 

A  "  neutral  "  solution  is  one  in  which  (H)  at  21°  C.  = 
io-7. 

An  "  acid  "  solution  is  one  in  which  (H)  at  21°  C.  is 
greater  than  io~7. 

An  "  alkaline  "  solution  is  one  in  which  (H)  at  21°  C.  is 
less  than  io~7. 

Acids  differ  markedly  in  the  degree  to  which  they  are 
ionised  in  solution.  "  Strong  "  acids,  like  HC1  or  HNO3, 


16  THE    PROPERTIES   OF   SOLUTIONS.  [CH.    I. 

are  freely  ionised  ;  whilst  "  weak  "  acids,  like  acetic  acid, 
are  only  feebly  ionised. 

By  electrical  measurements  of  the  conductivity  of  the 
solution  it  has  been  shewn  that  o.i  N.HC1  is  ionised  to  the 
extent  of  84  per  cent,  at  i8°C.  If  it  were  completely 
ionised  there  would  be  o-  i  gm.  of  hydrion  per  litre.  As  it  is 
only  partially  ionised, 

(H)  is  o-i  x  —  =  0-084  =  8-4  x  io-2  at   18°  C. 
100 

Similarly,  o.iN.  acetic  acid  is  only  dissociated  to  the 
extent  of  1*36  per  cent.  So  in  this  case 

(H)  =  o-i  x  — ?-  =  0-00136  =  1-36  x  io~3. 
100 

This  method  of  expressing  the  hydrogen  ion  concentra- 
tion is  not  convenient.  It  is  preferable  to  adopt  the  nota- 
tion of  Sorensen,  who  introduced  the  symbol  PH  to  denote 
the  "  hydrogen-ion-exponent."  PH  is  the  logarithm  to  the 
base  io  of  (H),  the  negative  sign  being  omitted.  In  other 
words 

PH  =  -logio   (H). 

A  few  examples  should  make  its  meaning  clear. 
o-iN.HCl  has  (H)  =  8-4  x  io~2.     Now  Iog10  8-4  =  0-92. 
So    8-4  x  io-2  =  io°'92-2  =  IO-1'08.     So  PH  =  1-08. 
o-iN.   acetic  acid   has 

(H)  =  1-36  x  io-3  =  io°-133-3  =  io-2-867. 
So  PH  =  2-867. 

It  will  be  observed  that  PH  decreases  as  the  acidity  in- 
creases. Also  that  if  (H)  is  doubled,  PH  is  not  halved,  but 
only  decreased  by  0-301,  since  Iog102  =  0-301. 

It  is  important  to  note  that  the  PH  of  a  solution  cannot 
be  determined  by  the  ordinary  method  of  titration.  Let 
us  consider  the  case  of  o-i  N.HC1  and  o-i  N.  acetic  acid. 
If  these  be  titrated  with  o-i  N.NaOH  until  they  each  give 
a  pink  with  phenol  phthalein,  io  cc.  of  each  acid  will 
require  exactly  ic  oc.  of  the  alkali,  and  will  therefore 


CH.    I.]  "  BUFFERS."  17 

have  apparently  the  same  acidity.  But  actually  the 
hydrochloric  acid  has  an  (H)  over  60  times  greater  than 
the  acetic  acid.  The  reason  for  this  is  that  the  acetic 
acid  is  only  very  slightly  ionised,  the  amount  ionised 
being  a  certain  proportion  of  the  total  acid  present. 
As  soon  as  the  ionised  part  has  been  removed  by  the 
addition  of  a  base,  a  further  fraction  of  the  previously  un- 
dissociated  acid  is  ionised.  This  process  is  repeated  with 
further  additions  of  alkali  until  the  whole  of  the  acid 
originally  present  has  become  dissociated,  and  its  hydrogen 
ions  have  united  with  the  hydroxyl  ions  of  the  base.  The 
next  trace  of  added  alkali  reacts  with  the  indicator  to  give 
a  pink  colour.  Titration  therefore  only  gives  us  an  index 
of  the  capacity  of  the  solution  to  neutralise  acids  or  alkalies  ; 
it  does  not  give  us  information  concerning  the  potential  of 
the  hydrogen  ions,  i.e.  the  PH. 

"Buffers."  A  single  drop  of  0-02  N.HC1  added 'to  a 
litre  of  pure  water  at  18°  C.  would  cause  a  change  in  PH 
from  7-07  to  about  6.  A  trace  of  diffusible  alkali  from  a 
glass  bottle  might  change  the  PH  to  8,  or  even  higher, 
whereas  exposure  to  the  CO2  of  the  air  might  cause  a  drop 
to  about  6.  Thus  it  is  extremely  difficult  to  maintain  any 
constancy  of  PH  in  such  a  solution.  But  with  certain 
substances  present  the  addition  of  a  small  amount  of  acid 
or  alkali  causes  only  a  minimal  change  in  PH.  Such 
substances  are  called  "  Buffers."  Various  solutions  are 
used  for  this  purpose,  such  as  phosphates,  citrates,  borates, 
and  acetates.  Let  us  consider  the  case  of  a  solution  of 
sodium  acetate,  to  which  is  added  a  small  amount  of 
hydrochloric  acid.  Both  substances  are  freely  dissociated 
so  that  the  following  ions  are  originally  present,  Na, 
(CH3.COO,  H,  Cl.  Now  acetic  acid  is  a  weak  acid,  which 
means  that  CH3.COO  and  H  ions  can  exist  together  only  in 
very  low  concentrations.  We  therefore  get 

Na  +  CH3.COO  +  H  +  Cl  =  CH3.COOH  +  Na  +  Cl. 

Thus  the  H  ions  of  the  added  hydrochloric  acid  nearly 
disappear,   owing  to  the  presence  of  the  buffer  sodium 


18  THE    PROPERTIES    OF   SOLUTIONS.  [CH.    I. 

acetate.     It  must  be  noted  that  they  do  not  all  disappear, 
for  some  of  the  acetic  acid  formed  is  dissociated  into  H  and 

CHg.COO. 

In  the  animal  body  the  proteins,  sodium  bicarbonate, 
and  phosphates  all  function  as  buffers,  and  help  to  maintain 
a  constancy  in  the  hydrogen  ion  concentration  of  the  tissue 
fluids. 

The  effect  o!  dilution  on  (H).  With  a  weak  acid  of 
the  type  HA,  the  extent  of  dissociation  is  governed  by  the 
equation 

K  .....  <„ 


(HA)  is  the  concentration  of  the  undissociated  mole- 
cules per  litre,  and  K  is  the  "  Dissociation  constant  "  of 
the  acid. 

Since  (H)  =  (A),  we  can  write  this 


(H)2  =  K(HA)     or    (H)  =  \/K(HA). 

This  indicates  that  if  a  solution  of  a  weak  acid  be  diluted 
four  times  (H)  is  halved;  if  it  be  diluted  16  times  it  is 
reduced  to  one-fourth. 

In  the  presence  of  any  considerable  amount  of  the 
sodium  salt,  the  effect  of  dilution  is  quite  different. 

We  can  write  equation  (i)  in  the  following  form  : 

__  K(HA) 

~~ 


The  sodium  salts  of  weak  acids  are  very  freely  dis- 
sociated, so  that  there  is  a  relatively  high  concentration  of 
A  ions  in  the  solution  of  the  mixture.  From  equation  (2) 
it  will  be  seen  that  an  increase  of  (A)  must  cause  a  decrease 
in  (H).  The  dissociation  of  the  weak  acid  being  thus 
depressed  it  follows  that  practically  all  the  acid  is  present 
in  the  undissociated  form,  so  that  we  can  assume 
that  (HA)  =  (acid).  Further,  practically  all  the  free  ions 
arise  from  the  dissociation  of  the  sodium  salt,  so  that  (A)  =*. 
(Sodium  salt.) 


CH.  I.]         HYDROGEN  ION  CONCENTRATION.  19 

We  can  therefore  write  equation  (2)  as 

K  (acid)  / 


(Sodium  salt) 

Since  the  sodium  salt  is  not  fully  dissociated,  except 
in  high  dilutions,  it  is  more  correct  to  write  it 

(U)  -          K  (acid)  (A\ 

a  (Sodium  salt) 

where  a  is  the  degree  of  dissociation  of  the  salt. 

It  follows  from  this  that  the  (H)  of  such  a  mixture  is 
mainly  conditioned  by  the  relative  concentrations  of  the 
acid  and  of  its  salt,  and  is  only  very  slightly  affected  by 
dilution,  which  does  not  alter  the  relative  concentrations. 
This  is  of  considerable  importance,  since  a  large  number  of 
physiological  fluids  can  be  regarded  as  mixtures  of  weak 
acids  with  their  sodium  or  potassium  salts,  and  so  suffer 
little  change  in  (H)  on  dilution. 

The  determination  of  the  hydrogen  ion  concentration. 

The  most  accurate  method  is  an  electrical  one,  involving 
expensive  and  intricate  apparatus.  It  is  too  complicated 
to  be  described  here.  A  valuable  method  that  does  not 
require  elaborate  apparatus  is  the  "  indicator,"  or  "  colori- 
metric  "  method. 

An  indicator  is  a  substance  that  varies  in  colour  tone  or 
in  depth  of  colour  with  the  PH  of  the  solution.  Each 
indicator  shows  a  colour  change  over  a  certain  range  of  PH. 
At  some  particular  PH  the  indicator  may  show  an  inter- 
mediate or  faint  tint.  The  solution  is  then  said  to  be 
"  neutral  "  to  this  indicator.  It  does  not  follow  that  the 
solution  is  neutral  in  the  strict  sense,  i.e.  (H)  =  (OH). 
The  PH  of  a  solution  "  neutral  to  phenol  phthalein  "  is 
about  9  ;  that  of  a  solution  "  neutral  to  methyl  orange  "  is 
about  4,  the  (H)  in  the  latter  case  being  100,000  times 
greater  than  in  the  former. 

The  method  adopted  for  the  determination  of  PH  by 
indicators  is  to  take  standard  solutions  of  certain  substances 


20 


THE    PROPERTIES    OF   SOLUTIONS. 


[CH.    I. 


which  can  be  mixed  in  various  proportions  to  give  a  series 
of  solutions  of  a  known  PH>  which  have  been  accurately 
determined  by  the  electrical  method.  A  given  amount  of  a 
suitable  indicator  is  added  to  a  measured  volume  of  the 
fluid,  and  also  to  equal  volumes  of  the  standard  test 
solutions,  contained  in  tubes  or  vessels  as  uniform  as 
possible.  The  solutions  that  give  exactly  the  same  tints 
have  the  same  hydrogen  ion  concentration,  provided  that 
this  concentration  is  in  the  range  of  the  indicator  employed. 
The  results  are  not  as  accurate  as  the  electrical  method, 
owing  to  the  difficulty  of  exactly  matching  the  tints  and 

SOURCE  OF  LIGHT. 


Water. 


Coloured 
fluid. 


B 

Standard 


Indicator. 


Ground 
glass 


EYE. 

Fig.  3.     Plan  of  arrangement  of  tubes  in  Cole  and  Onslow's  Comparator. 


also  of  the  effect  of  proteins,  salts,  and  other  substances 
on  the  colour  developed.  If  the  fluid  be  coloured  it  is 
obvious  that  this  simple  method  can  only  give  very  approxi- 
mate results.  Walpole  overcame  the  difficulty  by  viewing 
the  (standard  solution  +  indicator)  through  a  layer  of  the 
coloured  fluid  of  the  same  depth  as  that  of  the  (coloured 
fluid  +  indicator).  A  special  instrument  was  devised  for 
this  purpose.  Hurwitz,  Meyer  and  Ostenberg  used 
Walpole 's  principle,  but  employed  test  tubes  held  in  a  box 


CH.    I.] 


DETERMINATION   OF   PH. 


21 


or  "  comparator."  Cole  and  Onslow  somewhat  improved 
this  by  using  a  comparator  containing  three  pairs  of 
tubes,  the  arrangement  being  diagramatically  shown  in 
fig.  3.  The  addition  of  a  ground  glass  plate  fixed  to  the 
comparator  between  the  eye  and  the  tubes  has  been  found 
by  the  author  very  much  to  improve  the  apparatus,  slight 
differences  in  colour  being  readily  detected. 

The  standard  solutions  taken  are  such  that  the  appear- 
ance seen  through  Y  is  either  intermediate  between  that 
seen  through  X  and  Z,  or  identical  with  one  of  them.  The 
colour  changes,  and  the  ranges  of  the  most  useful  indicators 
are  given  below  in  Table  I.  Certain  other  interesting  data 
are  presented  in  chart  form  in  Table  II. 


Fig  4.     Cole  and  Onslow's  Comparator. 

The  author  can  very  strongly  recommend  the  new 
sulphone-phthalein  indicators*  introduced  by  Mansfield 
Clark,  Lubs  and  Acree.  For  further  information  on  the 
subject  of  the  colorimetric  method  of  determination  of  PH 
the  student  is  referred  to  an  important  series  of  papers  by 
Clark  and  Lubs,  "  Journal  of  Bacteriology,"  Baltimore, 
Vol.  II.,  pp.  i,  109  and  191  (1917). 


*  The  Cooper  Laboratory  for  Economic  Research,  Watford,  Herts., 
has  undertaken  the  manufacture  of  the  sulphone-phthalein  indicators. 
They  can  be  obtained  in  convenient  standardised  solutions,  or  in  the  solid 
form,  either  direct  or  through  Messrs.  Baird  and  Tatlock  and  other  agents. 


22  THE    PROPERTIES    OF    SOLUTIONS.  [CH.    I. 

TABLE    I. 
Indicators. 

Those  printed  in  heavy  type  are  the  most  useful   for 
ordinary  work. 


TRADE 
NAME 

CHEMICAL 
COMPOSITION 

RANGE 
OF  PH 

COLOUR 
CHANGE, 
Acid—  alkaline. 

i.  Methyl  violet 

o-i    to     3-2 

Green-blue 

2.  Thymol  blue 

Thymol    -    sulphone    - 
phthalein 

1-2     to     2-8 

Red-yellow 

3.  Toepfer's  re- 
agent 

Di  -  methyl  -  amino  - 
azo-benzene 

2-9    to    4-2 

Red-yellow 

4.  Brom-phenol- 
blue 

Tetra  -  brom  -  phenol  - 
sulphone-phthalein 

2-8    to    4-6 

Yellow-blue 

5.  Methyl 
orange 

p  -  benzene  -  sulphonic  - 
acid-azo-di-methyl- 
aniline 

3-i    to    4-4 

Red-yellow 

6.  Congo  red 

3-0    to    4-5 

Blue-red 

7.  Methyl-red 

p    -    dimethyl   amino   - 
azo   -  benzene  -   o  - 
carbonic  acid 

4-4    to    6-0 

Red-yellow 

8.  Brom-cresol- 
purple 

Di  -  brom  -  o  -  cresol  - 
sulphone-phthalein 

5-2    to    6-8 

Yellow-purple 

9.  Litmus 

5'4    to    7-8 

Red-blue 

10.  Brom-thymo) 
blue 

Di  -  brom  -  thymol  - 
sulphone  -  phthalein 

6-0    to    7-6 

Yellow-blue 

ii.  Neutral  red 

6-8    to    8-0 

Red-yellow 

12.  Phenol-red 

Phenol-sulphone- 
phthalein 

6-8    to    8-4 

Yellow-red 

13.  Cresol-red 

o  -  cresol  -  sulphone  - 
phthalein 

7-2    to    8-8 

Yellow-red 

14.  Thymol-blue 

Thymol  -  sulphone  - 
phthalein 

8-0    to    9-6 

Yellow-blue 

15.  Phenol 
phthalein 

Phenol  phthalein 

8-3  to  io-o 

Colourless-red 

1  6.  Thymol- 
phthalein 

Thymol-phthalein 

8-3  to  10-5 

Colourless-blue 

CH.    I.] 


INDICATORS. 


23 


Preparation  of  solutions  * 

Alcoholic  solutions  are  prepared  by  dissolving  the  solid  in 
alcohol  and  diluting  with  water.   . 

(i)  o-oi  to  0-05  per  cent,  in  water.  (2)  0-04 
per  cent,  in  water  by  diluting  10  cc.  of  the  stock 
(1-2  per  cent.)  solution  with  290  cc.  water. 

(3)  0-02  per  cent,  in  50  per  cent,  alcohol. 
(4)  Hke  (2). 

(5)  o-oi  per  cent,  in  water.  (6)  0-02  per  cent. 
in  water. 

(7)  0-02  per  cent,  in  60  per  cent,  alcohol. 
(8)  as  (2). 

(9)  Strong  aqueous  solution,  dialysed  against 
distilled  water. 

(10)  As  (2).      (n)  o-oi   per  cent,  in   50  per 
cent,  alcohol. 

(12)  0-02  per  cent,  in  water,  by  diluting 
10  cc.  of  the  stock  (0-6)  per  cent,  solution  with 
290  cc.  water.  (13)  as  (12). 

(14)  Same  as  solution  (2).  (15)  0-05  per  cent. 
in  50  per  cent,  alcohol.  (16)  0-04  per  cent,  in  50 
per  cent,  alcohol. 

Volume  required.  In  most  cases  ten  drops 
to  10  cc.  of  the  solution  are  about  right.  But 
the  amount  varies  with  the  range,  colour  of  solution, 

etc.     Thus  12  drops  of  no.  (12)  maybe  required  Fif;5-  Bottleand 

J  ^  Dreyer  s  Drop- 

at  PH  =  6-9,  and   only  5  drops  at  PH  =  8-0.     It      ping      Pipette 

is    essential   that    exactly  the  same  amount  be 

added  to  the  measured  volume  of  the  fluid  and  to 

the  same  measured  volumes  of  the  standard  solutions.    The  most 

convenient  and  accurate  method  of  adding  the  drops  is  to  have  the 

bottle  of  indicators  fitted  with  rubber  corks  pierced  with  Dreyer's 

dropping  pipettes  (fig.  5). 

Colour  filters  for  dichroic  indicators.      Brom-phenol  blue  and 
brom-cresol  purple  are  dichroic.    To  get  reliable  results,  especially 


*  Concentrated  standardised  solutions  can  be  obtained  from  the  Cooper 
Laboratory  for  Economic  Research,  Watford,  Herts. 


24  THE    PROPERTIES    OF    SOLUTIONS.  [CH.    I. 

with  turbid  fluids,  it  is  necessary  to  compare  the  solutions  by 
using  a  source  of  light  from  which  the  blue  rays  have  been 
screened  off.  This  can  be  done  by  painting  a  piece  of  trans- 
parent tracing  paper  with  a  strong  acid  solution  of  phenol  red, 
prepared  by  mixing  one  part  of  the  stock  0-6  per  cent,  solution 
with  one  part  of  the  standard  0-2  M  acid  potassium  phosphate. 
The  paper,  while  still  wet,  is  pinned  across  the  front  of  a  box 
containing  one  or  two  powerful  carbon  filament  lamps.  The  ex- 
amination should  be  conducted  in  a  dark  room,  or  the  external 
light  should  be  cut  off  by  using  a  dark  cloth. 

Another  method  is  to  take  an  unexposed  photographic  plate, 
fix  it  in  hypo  in  a  dark  room,  wash  for  some  hours  in  running  water, 
stain  by  immersion  in  the  dye,  drying  and  mounting  a  piece  on  the 
comparator  on  the  side  towards  the  light,  in  place  of  the  ground 
glass  screen. 

Standard  solutions  of  definite  PH. 

The  most  convenient  sets  of  solutions  that  have  been 
worked  out  are  those  of  CJark  and  Lubs.  A  constant 
volume  (5occ.)  of  a  standard  solution  of  acid  potassium 
phthalate,  acid  potassium  phosphate  or  of  boric  acid  is 
measured  into  a  200  cc.  measuring  flask.  A  given  amount 
(x  cc.)  of  standard  NaOH  or  HC1  is  then  added,  and  the 
volume  brought  to  the  mark  with  distilled  water.  The 
PH  obtained  with  the  different  solutions  are  given  in  the 
tables  below.  If  intermediate  points  are  desired,  they  can 
be  obtained  from  curves  drawn  from  the  points  given. 

Preparation  of  solutions. 

0-2  M  acid  potassium  phthalate.  Dissolve  40-828  gm.  in  dis- 
tilled water  and  make  up  to  I  litre.  The  salt  should  be  recrystallised 
from  distilled  water  and  dried  at  110°  C.  for  some  hours. 

0-2  M  acid  potassium  phosphate.  Dissolve  27-231  gm..in  dis- 
tilled water  and  make  up  to  i  litre.  The  salt  should  be  recrystallised 
from  distilled  water  and  dried  at  110°  C.  for  some  hours. 

0-2  M  Boric  Acid  in  0-2  KCl.  Dissolve  12-4048  gm.  of  air 
dried  boric  acid  and  14-912  gm.  pure  ignited  KG  in  distilled  water 
and  make  up  to  i  litre. 


CH.    I.] 


STANDARD    SODA. 


25 


Sodium  Hydroxide. 

Dissolve  100  grams  of  the  best  NaOH  in  100  cc.  of  distilled 
water  in  an  Erlenmeyer  flask  of  resistance  glass.  Cover  the  mouth 
of  the  flask  with  tin  foil,  and  allow  the 
solution  to  stand  over-night  till  the  carbon- 
ate has  mostly  settled.  Cut  a  hardened 
filter  paper  to  fit  a  Buchner  funnel.  Treat 
it  with  warm  strong  [i :  i]  NaOH  solution. 
Decant  the  soda  and  wash  the  paper  first 
with  absolute  alcohol,  then  with  dilute 
alcohol,  and  finally  with  large  quantities 
of  distilled  water.  Place  the  paper  on  the 
Buchner  funnel  and  apply  gentle  suction 
until  the  greater  part  of  the  water  has 
evaporated.  Now  pour  the  concentrated 
alkali  upon  the  middle  of  the  paper,  spread 
it  with  a  glass  rod,  and  filter  under  suction. 
The  clear  solution  is  now  diluted  quickly 
with  cold  distilled  water,  that  has  been 
recently  boiled  to  remove  C02,  to  make 
approximately  N.  NaOH ;  10  cc.  of  this 
is  withdrawn  and  roughly  standardised 
against  N.  HC1.  It  is  then  diluted  till  it  is 
approximately  0-2  N  with  CO2-free  water 
and  the  solution  poured  into  a  paraffined 
bottle,  to  which  a  burette  and  soda-lime 
guard  tubes  have  been  attached  (see  fig.  6). 
The  solution  is  then  accurately  standardised  against  weighed  amounts 
of  the  pure  acid  potassium  phthalate.  To  do  this  accurately  weigh  up 
about  1-5  gms.  of  the  salt,  dissolve  in  about  30  cc.  of  distilled  water, 
add  phenol  phthalein  and  titrate  with  the  alkali  till  a  faint  but  dis- 
tinct and  permanent  pink  is  developed.  A  current  of  CO2-free  air 
should  be  blown  through  the  solution  during  the  titration.  The 
apparatus  shewn  in  fig.  35  is  convenient  for  this  purpose. 

If  p  be  the  exact  weight  of  the  phthalate  taken,  and  s  the 
volume  of  soda  required,  the  normality  of  the  soda  is 

T-OOO  x  p 


Fig.  6.  Paraffined  bottle 
(A)  for  storing  standard 
alkali.  B  and  C  are 
soda  lime  tubes.  The 
burette  is  filled  by 
sucking  at  C. 


=  a. 


204-14  x  s 


THE  PROPERTIES  OF  SOLUTIONS. 


[CH.  I. 


Instead  of  using  x  cc.  of  0-2  N,  the  amount  of  the  standardised 
soda  that  must  be  employed  is 


X  X   O-2 


CC. 


It  is  convenient  to  label  the  bottle  with  the  factor 


0-2 


0-2  N  Hydrochloric  acid.  This  can  be  prepared  from  a  freshly 
distilled  20  per  cent,  solution,  and  standardised  against  the  standard 
soda,  using  methyl  red  as  the  indicator. 

Series  A.     50  cc.  0-2  M.  acid  potassium  phthalate. 
x  cc.  0-2  N.  HC1. 
Diluted  to  200  cc. 

(Thymol  blue. 


Indicators  recommended 


j  Brom-phenol  blue. 


PH 

% 

PH 

X 

PH 

X 

2-2 

4670 

2-9 

22-80 

3-6 

5'97 

2-3 

42-50 

3'0 

20-32 

37 

4-30 

2-4 

39-60 

3'i 

17-70 

3'8 

2-63 

2'5 

37-00 

3'2 

14-70 

3'9 

I  -00 

2-6 

32-95 

3'3 

11-80 

27 

29-60 

3'4 

9-90 

2-8 

26-42 

3*5 

7-50 

Series  B.    50  cc.  0-2  M.  acid  potassium  phthalate. 


x  cc  0-2  N.  NaOH. 
Diluted  to  200  cc. 

Indicators  recommended 


Brom-phenol  blue. 
Methyl  red. 
Brom-cresol  purple. 


CH.    I.] 


STANDARD    SOLUTIONS. 


27 


PH 

M 

PH 

x 

PH 

x 

4-0 

0-40 

4-8 

1770 

5-6 

39-85 

4'1 

2-2O 

4'9 

20-95 

5-7 

41-90 

4-2 

370 

5'0 

23-85 

5-8 

43-oo 

4-3 

5-17 

5'1 

27-20 

5'9 

44-55 

4'4 

7-50 

5-2 

29-95 

6-0 

45-45 

4-5 

9-60 

5-3 

32-50 

6-1 

46-20 

4-6 

12-15 

5-4 

35-45 

6-2 

47-00 

47 

14-60 

5-5 

37-70 

6-3 

48-10 

Series  C.    50  cc.  0-2  M.  acid  potassium  phosphate. 
x  cc.  0*2  N.  sodium  hydroxide. 
Diluted  to  200  cc. 

Brom-cresol  purple. 
Indicators  recommended     •]  Brom- thymol  blue. 

Phenol  red. 


PH 

% 

PH 

% 

PH 

% 

5-8 

3-72 

6-6 

17-80 

7*4 

39-50 

5-9 

470 

67 

21  -OO 

7-5 

41-20 

6-0 

570 

6-8 

23-65 

7-6 

42-80 

6-1 

7-40 

6-9 

26-50 

7'7 

44-20 

6-2 

8-60 

7-0 

29-63 

7-8 

45-20 

6-3 

10-19 

7-1 

32-50 

7-9 

46-00 

6-4 

12-60 

7-2 

35-oo 

8-0 

46-80 

6-5 

16-00 

7-3 

37-40 

Series  D.     50  cc.  0-2  M.  boric  acid  in  0-2  M.  potassium  chloride. 
x  cc.  0-2  N.  sodium  hydroxide. 
Diluted  to  200  cc. 

(Cresol   red. 


Indicators  recommended 


(Thymol  blue. 


28 


THE    PROPERTIES   OF   SOLUTIONS. 


[CH.    I. 


PH 

% 

PH 

X 

PH 

X 

PH 

X 

7-8 

2-61 

8-4 

8-50 

9-0 

21-30 

9-6 

36-85 

7*9 

3-30 

8-5 

10-40 

9-1 

24-30 

97 

39-00 

8-0 

3'97 

8-6 

I2-OO 

9-2 

26-70 

9-8 

40-80 

8-1 

4-80 

87 

I4-30 

9*3 

29'95 

9-9 

42-50 

8-2 

5'9° 

8-8 

16-30 

9-4 

32-00 

10-0 

43-90 

8-3 

7'3o 

8-9 

IQ'OO 

9'5 

34-50 

• 

Series  E.     Sodium  acetate  and  acetic  acid. 

The  following  series  is  given  as  being  convenient  for  certain 
experiments.  It  should  be  noted  that  the  PH  of  the  solutions  is 
only  very  slightly  changed  by  considerable  dilution  with  water.* 

Preparation  of  Solutions. 

N.  acetic  acid  is  prepared  by  titration  against  N.  soda.  0-2  N. 
acetic  acid  is  prepared  from  this  by  diluting  200  cc.  to  1000  cc.  with 
distilled  water.  6-2  N.  sodium  acetate  is  prepared  by  mixing  200  cc. 
of  the  N.  acetic  acid  with  200  cc.  of  the  N.  soda  employed  and 
diluting  to  1000  cc.  with  distilled  water. 

Take  x  cc.  of  the  sodium  acetate,  and  add  (IQ-X)  cc.  of  the 
0-2  N.  acetic  acid. 


PH 

X 

(10-*) 

PH 

X 

(10-*) 

3'8 

1*2 

8-8 

4-8 

5*95 

4-05 

3-9 

i-5 

8-5 

4'9 

6-5 

3-5 

4-0 

1-8 

8-2 

5'° 

7-0 

3'0 

4-1 

2-2 

7-8 

5'1 

7-45 

2-55 

4-2 

2-65 

7-35 

5-2 

7-85 

2-15 

4'3 

3-1 

6-9 

5-3 

8-25 

I"75 

4-4 

37 

6-3 

5-4 

8-5 

1'5 

4-5 

4-25 

575 

5-5 

8-8 

1*2 

4-6 

4-8 

5-2 

5-6 

9-05 

0-95 

47 

5-4 

4-6 

57 

9-25 

075 

*  See  page  18. 


CH.    I.]  DETERMINATION    OF   PH.  29 

8.    The  determination  of  the  PH  of  urine. 

Apparatus  and  reagents  required. 

(1)  A  number  of  clean,  dry  test-tubes  of  thin  clear  glass  and  of 
uniform  bore,     f  inch  is  a  suitable  external  diameter. 

(2)  A  comparator  for  holding  the  tubes.     This  is  shown  in 
figure  4. 

(3)  A  series  of  buffer  solutions  of  known  PH  prepared  according 
to  directions  given  above.     It  is  convenient  to  have  a  series  of  these 
prepared  and  contained  in  bottles  fitted  with  a  rubber   stopper, 
through  which  passes  the  stem  of  a  5  cc.  pipette. 

(4)  Solutions  of  appropriate  indicators  (see  page  23).     Methyl 
red,  brom-cresol-purple,  brom-thymol  blue  cover  the  range  of  the 
majority  of  specimens  of  urine. 

(5)  A  screen  to  cut  out  blue  rays  when  using  brom-cresol-purple 
(see  page  23). 

Method.  To  5  cc.  of  the  filtered  urine  add  5  drops  of  methyl  red. 
If  the  mixture  is  red,  the  PH  is  in  the  neighbourhood  of  5.  If  it  is 
yellow,  the  PH  is  nearer  6.  In  the  latter  case  treat  another  5  cc.  with 
5  drops  of  brom-cresol-purple.  A  deep  purple  tint  suggests  that 
the  PH  is  higher  than  6,  in  which  case  it  may  be  necessary  to  use 
brom-thymol-blue.  Having .  roughly  obtained  the  range  and  the 
necessary  indicator,  place  about  5  cc.  of  the  specimen  into  two  of  the 
special  tubes  and  place  them  in  the  holes  marked  2  and  6.  Into  a 
tube  in  4  place  some  distilled  water. 

Measure  exactly  5  cc.  of  the  urine  into  another  tube,  and  to  it  add 
5  drops  of  the  indicator  (measured  with  a  Dreyer's  dropping  pipette, 
fig.  5).  Mix  by  rotating  the  tube  between  the  palms  of  the  hands, 
and  place  the  tube  in  the  hole  marked  3.  Measure  5  cc.  of  one  of  the 
buffer  solutions  into  a  tube,  add  5  drops  of  the  indicator  mix,  and 
place  the  tube  in  the  hole  marked  i.  Hold  the  comparator  to  the 
source  of  light  with  the  ground  glass  screen  towards  the  observer 
and  note  the  appearance  opposite  the  slots  x  and  y.  It  will  then 
be  ascertained  whether  the  buffer  chosen  is  acid  or  alkaline  to  the 
urine.  In  either  case  another  buffer  solution  must  be  taken,  the 
indicator  added,  and  the  tube  placed  in  the  hole  5,  and  the  tubes 


30  THE    PROPERTIES    OF   SOLUTIONS.  [CH.    I. 

examined  again.  This  procedure  must  be  repeated  until  two  solu- 
tions are  found  of  such  a  PH  that  the  colour  as  seen  through  y  is 
intermediate  between  those  seen  through  x  and  z,  or  that  through  y 
is  identical  with  one  of  them.  The  PH  of  the  solutions  finally 
employed  should  not  differ  by  more  than  O'i. 

NOTE. — In  measuring  the  indicator  solutions  it  is  essential  to  hold  the 
dropping  pipette  vertical,  to  ensure  the  delivery  of  equal  drops. 


H.    Ampholytes  or  amphoteric  electrolytes. 

These  are  substances  which  can  function  as  acids  by 
forming  salts  with  bases,  and  also  as  bases  by  forming  salts 
with  acids.  The  amino  acids,  such  as  glycine,  are  examples. 
Glycine  can  form  a  sodium  salt,  CH2.NH2.COONa,  and 
also  a  hydrochloride,  HC1.H2N.CH2.COOH.  In  strong 
acids  it  behaves  as  a  base  ;  in  strong  alkalies  as  an  acid.  In 
neutral  solutions  it  is  a  feeble  electrolyte,  and  is  partially 
dissociated  into  H  and  a  negative  ion  (anion). 

H2N.CH2.COOH  ±  Z^  H2N.CH2COO  +  H...(i) 
and  partially  into  OH  and  a  kation. 
HO.H3N.CH2.COOH  < ~  HO  +  H3N.CH2.COOH...(2) 

If  a  strong  acid,  such  as  HC1,  be  added  the  dissociation  (i)  is 
decreased,  in  the  same  way  as  the  dissociation  of  all  weak  acids  is 
decreased  by  an  increase  in  the  hydrogen-ion  concentration.  On 
the  other  hand  the  number  of  kations  formed  is  increased,  since  such 
a  salt  as  glycine  hydrochloride  is  freely  dissociated. 

If  a  strong  base,  like  NaOH,  be  added  the  dissociation  (2)  is 
depressed,  and  there  is  an  increase  in  the  number  of  anions  of  the 
ampholyte,  due  to  the  free  dissociation  of  the  sodium  salt  that  is 
formed. 

For  every  ampholyte  there  is  some  particular  concen- 
tration of  hydrogen  ions  at  which  the  dissociation  (i)  is 
equal  to  the  dissociation  (2).  This  is  known  as  the  "  iso- 
electric  point."  Since  the  proteins  are  ampholytes,  the 


CH.    I.]  AMPHOLYTES.  31 

conditions  of  a  substance  at  its  iso-electric  point  are  of  some 
interest.     They  are  : 

1 i )  The  sum  of  the  anions  and  kations  is  at  a  minimum. 

(2)  The  concentrations  of  the  anions  and  kations  are 
equal. 

(3)  If  an  ampholyte  be  added  to  a  solution,  whose  [H] 
is  greater  than  its  iso-electric  point,  it  functions  as  a  base, 
and  therefore  decreases  the  [H]  of  the  solution.     If  it  be 
added  to  a  solution,  whose  [H]  is  less  than  its  iso-electric 
point,  it  functions  as  an  acid.     If  the  (H)  of  a  solution  is 
no1"  altered  by  addition  of  an  ampholyte,  then  the  (H)  of 
the  solution  must  be  equal  to  the  iso-electric  point  of  the 
ampholyte. 

(4)  The  solubility  of  ah  ampholyte  is  at  a  minimum  at 
its  iso-electric  point.    If  a  colloid  the  ampholyte  flocks  most 
readily  at  this  point.     (See  Ex.  6.) 

(5)  At  its  iso-electric  point,  a   colloid  is  electrically 
neutral. 

The  significance  of  the  iso-electric  points  of  various 
enzymes  is  discussed  on  p.  184.  The  iso-electric  points  of 
certain  substances  of  physiological  interest  are  shewn  in 
Table  II.,  p.  32. 


32 


CHART  OF  INDICATORS  AND  REACTIONS. 


[CH.  I. 


TABLE  II. 


RANGE 

OF          REACTIONS  OF                        OPTIMUM                         ISOELECTRIC 

p            ^RINCIPAL                 FLUIDS                              REACTIONS                              POINTS                 p 

«       INDICATORS                                                                                                                                                .» 

z 

IU 

01 

-J 

—  . 

H 

X 

a 

a 

9- 

p 

, 

_ 

- 



9 

o 

•z. 

a 



W 

I 

0 
g 

Q 

a: 

(Pancreatic  juice  (8-3) 
^Intestinal  contents  (8-3) 

8- 

•  s 

9! 
H 

^ 



-»Trypsin  on  fibrin  (8-0)    - 



8 

o: 
j 

% 

UJ 

->Erepsin  (7-8) 

Q 

3i 

u 

UJ 

i 

-Blood  (7-4) 

X 

d 

THYMOL  BI 

-Human  mi.lk  (7.1) 

• 

-Histidine  (7-2) 

7 

7" 

? 

a. 

-Saliva  (6.9) 
—  ^Cow's  milk  (6-7) 

(Maltase  (6-7) 
Ptyalin  (6-7) 
Trypsin  on  casein  (6-7) 

xAlanine  (6-7) 
—  Oxyhxmoglohin  (6-7) 
^Glycine  (6-6) 

£ 

3 

a 

i 

K 

6- 

;s 

QQ 

-Urine  (6-0) 

- 



6 

U} 

u 

Q 
UJ 

xTyrosine  (5-41) 

Di 

CC 

<  Serum  globulin  (5-4) 

5- 

£ 

-J 

I 
E- 

-Infants'  gastric  juice  (5-0)- 

—Protease  of  Taka-diastase 
(5-1) 

\JSerum  albumin 
\    (denatured)  (5-4) 

5 

UJ 

—  Serum  albumin  (4-7) 

UJ 

- 

-Mnvertase  (4-5) 

rCasein  '(4-6) 
OGelatine  (4-6) 
xPhenyl-alanine  (4-48) 

CQ 

4- 

-0-0001  N.HC1  (4-01)       - 

— 



4 

0 

z 

01 

-0-001  N.  acetic  (3-87) 

I 

a 
& 

O 

-0-01  N.  acetic  (3-37) 

3- 

§ 
•  cc 



-0-001  N  .  HC1  (3-01) 
r-0-l  N.acetiaacid  (2.-S7) 

- 

-Aspartic  acid  (2-9) 

3 

§ 

-N.  acetic  (2-37) 

2- 

CC 

"  g 



-0-01N.HCI(2-.02) 

- 



2 

i- 

X 
h 

}  Adult  gastric  juice 
(0-9  to  1-6); 
0-1-  N.HC1{1-08> 

-Pepsin  (1-4) 

1 

CHAPTER   II. 
THE   PROTEINS. 

A.    Definition. 

Proteins  are  nitrogenous  compounds  found  in  the 
fluids  and  tissues  of  all  living  organisms.  Chemically,  they 
are  composed  of  a  number  of  amino  acids  (see  p.  67),  con- 
densed together  in  a  characteristic  way  so  that  the  whole 
molecule  is  generally  neither  very  acid  nor  very  basic. 
Their  chemical  properties  are  dependent  on  the  presence  of 
these  amino  acids.  Their  physical  properties  are  mainly 
due  to  the  fact  that  they  form  colloidal  solutions  (p.  i). 
The  percentage  composition  varies  very  considerably  in 
different  proteins.  The  following  can  be  taken  as  a  rough 
average  : — 

C    =53  per  cent. 

O   =  23     „       „ 

N   =  16     „       „ 

7     )>       » 
S    =    i 


100 


B.    Classification. 

It  is  not  possible  at  present  to  give  a  rational  scheme, 
for  we  have  not  sufficient  data  of  a  chemical  nature  by 
means  of  which  we  can  characterise  the  individual  proteins. 

The  classification  adopted  here  is  based  on  physical  and 
chemical  properties,  and  closely  follows  the  official  classi- 
fication of  the  American  Physiological  Society.  Where 
the  British  Society  uses  a  different  name  this  is  indicated 

by(B). 


34  THE    PROTEINS.  [CH.    II. 

1.  Protamines.    Basic  substances  forming  stable  salts  with 
mineral  acids,  and  containing  a  high  percentage  of  nitrogen.     On 
hydrolysis  they  yield  only  a  few  of  the  amino  acids,  and  these  are 
mainly  the  bases.     They  occur  in  the  heads  of  ripe  spermatozoa  and 
in  ova. 

2.  Histones.     Similar  to   the  protamines,   but  less  rich  in 
nitrogen  and  the  basic  amino  acids.     They  are,   however,  more 
basic  than  the  majority  of  the  proteins,  and  are  precipitated  by 
ammonia.     They  are  found  in  unripe  spermatozoa,  the  stroma  of 
red  blood  corpuscles,  and  in  lymphoid  tissue. 

3.  Albumins.    Soluble  in  distilled  water  and  coagulated  by 
boiling. 

4.  Globulins.     Insoluble  in  distilled  water,  soluble  in  dilute 
salt  solutions.     Coagulated  by  boiling. 

Groups  3  and  4  are  sometimes  known  as  "  native  "  proteins. 

5.  Glutelins.    Found  in  abundance  in  vegetables.     Insoluble 
in  neutral  solvents,  but  soluble  in  acids  and  alkalies. 

6.  Prolamines  [Gliadins  (B)].    Also  found  in  vegetables,  but 
distinguished  from  the  glutelins  by  their  solubility  in  75  per  cent, 
alcohol. 

7.  Albuminoids  [Scleroproteins  (B)].      Found  in  the  skeletal 
and  connective  tissues  of  animals.     They  are  characterised  by  their 
insolubility  in  most  reagents.     Examples  are  keratin,  elastin,  and 
collagen  (the  anhydride  of  gelatine). 

8.  Phospho-proteins.      Rich  in  phosphorus.     They  must  be 
carefully  distinguished  from  the  nucleoproteins.     Examples  are  the 
casein  [caseinogen  (B)]  of  milk  and  vitellin  of  egg  yolk. 

9.  Conjugated  Proteins.     Proteins  united  to  a  non-protein 
group. 

(i.)  Chromoproteins.  Protein  -f  pigment  molecule,  e.g.  haemo- 
globin. 

(ii.)  Glycoproteins  [Glucoproteins  (B)].  Protein  +  carbohy- 
drate group,  e.g.  mucin. 

(iii.)  Nucleoproteins.  Protein  +  nucleic  acid.  These  are  prob- 
ably indefinite  salts  of  nucleic  acid  with  proteins  (see 
p.  60). 


CH.    II.]  GENERAL    REACTIONS.  35 

10.    Hydrolysed  Proteins.     Formed  by  the  action  of  acids, 
alkalies  and  certain  enzymes  on  the  native  proteins. 

(i.)  Metaproteins.     Soluble  only  in  acids  and  alkalies, 
(ii.)  Proteoses  or  albumoses.    Soluble  in  water,  not  coagulated 

by  heat,  precipitated  by  ammonium  sulphate, 
(iii.)  Peptones.     Like   the  albumoses,  but  not  precipitated  by 

ammonium  sulphate. 

(iv.)  Polypeptides.     Simple    peptones,    formed    of    non-amino 
acids  only. 

C.    General  Reactions. 

For  the  following  reactions  use  egg-white  that  has  been  well  beaten  with 
6  times  its  volume  of  water  or  serum  that  has  been  diluted  ten  times  with  water. 

(i.)  The  proteins  give  certain  colour  reactions  (see 
pages  38  to  41). 

( 2 . )  They  are  precipitated  by  the  salts  of  the  heavy  metals. 
The  salts  that  are  most  used  are  lead  acetate,  mercuric 
chloride  and  nitrate,  ferric  chloride,  copper  sulphate,  and  zinc 
sulphate.  The  mechanism  of  the  precipitation  is  somewhat 
complex,  and  probably  varies  for  different  salts  and 
different  concentrations.  In  a  good  many  cases  it  seems 
to  be  due  to  the  adsorption  of  the  metallic  kation  by  the 
negatively  charged  colloidal  protein.  For  this  reason  the 
precipitation  is  best  obtained  when  the  reaction  of  the 
medium  is  somewhat  alkaline,  the  protein  then  being 
negatively  charged  (see  p.  n).  Also  the  precipitate  is 
often  soluble  in  acid.  It  is  often  soluble  in  an  excess  of  the 
-metallic  salt,  probably  because  the  charge  on  the  protein 
becomes  positive  owing  to  the  adsorption  of  the  excess  of 
positive  ions. 

9.  Treat  3  cc.  of  the  solution  with  a  few  drops  of  mercuric 
nitrate.  A  white  precipitate  is  obtained.  This  will  partially  or 
completely  dissolve  in  a  saturated  solution  of  sodium  chloride,  pro- 
vided that  the  solution  does  not  contain  free  acid.  The  solubility 
in  sodium  chloride  is  due  to  the  fact  that  mercuric  chloride  is 
formed.  This  salt  differs  from  the  nitrate  in  that  it  is  only  feebly 
dissociated. 


36  THE   PROTEINS.  [CH.   II. 

10.  Treat  3  cc.  of  the  protein  solution  with  ferric  chloride, 
drop  by  drop.     A  precipitate  is  formed  soluble  in  excess. 

11.  Treat  3  cc.  of  the  protein  solution  with  a  solution  of  lead 
acetate  or  basic  lead  acetate.     A  white  precipitate  is  formed. 

( 3 . )  The  proteins  are  precipitated  by  the  so-called ' '  alka- 
loidal  reagents"  These  include  phosphotungstic,  phos- 
phomolybdic,  ferrocyanic,  tannic,  picric,  metaphosphoric, 
and  sulphosalicylic  acids,  and  Briicke's  reagent  (potassio- 
mercuric  iodide). 

It  is  possible  that  the  precipitation  is  due  to  the  adsorp- 
tion of  the  complex  negative  ions  by  the  positively  charged 
colloidal  protein.  It  is  suggestive  that  the  substances  are 
only  effective  in  acid  solution,  in  which  the  proteins  are 
positively  charged.  The  precipitating  action  of  these  re- 
agents on  the  peptones  varies  somewhat.  As  a  rule  they 
are  not  so  readily  precipitated  as  the  albumins  and  globu- 
lins. 

12.  Treat  3  cc.  of  the  solution  with  two  or  three  drops  of  strong 
acetic  acid  and  two  drops  of  potassium  ferrocyanide.     A  white 
precipitate  is  formed.     Boil.     The  precipitate  does  not  dissolve. 

NOTES. — i.  Primary  proteoses  are  also  precipitated  by  ferrocyanic  acid, 
but  the  precipitate  produced  dissolves  on  warming  and  reappears  on  cooling 
(Ex.  55). 

2.  The  precipitate  and  fluid  often  become  coloured  blue-green  on  boiling. 
This  is  due  to  a  decomposition  of  the  hydroferrocyanic  acid  on  boiling  it  with 
certain  organic  substances,  such  as  proteins. 

13.  Acidify  some  of  the  solution  with  hydrochloric  acid  and 
add  a  few  drops  of  a  freshly  prepared  solution  of  tannic  acid,  or  of 
Almen's  reagent.     A  white  or  brown  precipitate  is  usually  formed. 

NOTE. — Almen's  reagent  consists  of  4  gm.  of  tannic  acid  in  8  cc.  of  strong 
acetic  acid  and  190  cc.  of  50  per  cent,  alcohol. 

14.  Treat  3  cc.  with  an  equal  volume  of  Esbach's  solution.    A 
yellowish  precipitate  is  formed. 

NOTE. — Esbach's  solution  is  prepared  by  dissolving  10  grms.  of  picric 
acid  and  10  grms.  of  citric  acid  in  water  and  making  the  volume  up  to  a  litre. 
It  is  extensively  used  for  the  determination  of  albumin  in  urine  (Ex.  421). 


CH.   II.]  PROTEIN    PRECIPITANTS.  37 

15.  Acidify  the  solution  with  dilute  hydrochloric  acid  and  add 
a   few   drops   of   potassio-mercuric  iodide   (Briicke's  reagent).     A 
white  precipitate  is  formed. 

NOTE. — Briicke's  reagent  is  prepared  by  dissolving  50  grms.  of  potassium 
iodide  in  500  cc.  of  distilled  water,  saturating  with  mercuric  iodide  (120  grms.), 
and  making  up  to  i  litre. 

1 6.  Acidify  a  few  cc.  of  the  solution  with  dilute  hydrochloric 
acid  and  add  a  few  cc.  of  a  2  per  cent,  solution  of  phosphotungstic 
acid.     A  white  precipitate  is  produced. 

17.  To  a  few  cc.  of  the  solution  add  a  drop  or  two  of  a  freshly 
prepared  25  per  cent,  solution  of  metaphosphoric  acid.     A  white 
precipitate  is  produced. 

NOTE. — Metaphosphoric  acid  (HPO3)  is  used  by  Folin  for  removing  pro- 
teins from  blood  and  urine  in  certain  quantitative  methods.  The  solution 
must  be  freshly  prepared,  as  on  standing  it  slowly  passes  over  into  ortho- 
phosphoric  acid  (H3PO4),  which  has  no  precipitating  action  on  proteins. 

18.  To  a  few  cc.  of  the  solution  add  a  small  amount  (a  large 
"  knife  point  ")  of  sulphosalicylic  acid,  or  a  drop  or  two  of  a  strong 
(20  per  cent.)  solution.     A  white  precipitate  is  obtained. 

NOTE. — The  reagent  is  of  considerable  value  for  the  detection  of  albumin 
in  urine.  (See  Ex.  371.) 

It  can  be  prepared  by  dissolving  13  grm.  salicylic  acid  in  20  grms.  H2SO4 
by  warming,  and,  after  cooling,  adding  67  cc.  of  water. 

(4.)  The  proteins  are  precipitated  by  strong  alcohol. 
The  albumins  and  globulins  are  rapidly  changed  by  alcohol 
at  room  temperature  into  modifications  that  are  insoluble 
in  water,  salt  solutions,  dilute  alkalies  or  acids.  That  is, 
they  are  coagulated. 

The  proteoses  and  peptones,  the  phosphoproteins,  and 
gelatin  are  precipitated  by  alcohol,  but  the  precipitate 
redissolves  in  water  or  dilute  alkalies. 

19.  Place  about  4  cc.  of  serum  in  a  test-tube  and  cool  to 
o°  C.  by  means  of  a  freezing  mixture.     Fill  the  tube  with  strong 
alcohol  that  has  previously  been  cooled  to  about  8°  C.,  and  mix.     A 
white  precipitate  of  the  proteins  is  formed.    Filter  at  once  and 
treat  the  precipitate  with  water.     It  dissolves. 


38  THE   PROTEINS.  [CH.    II. 

20.  Allow  a  few  drops  of  serum  to  fall  into  about  10  cc.  of 
strong  alcohol  at  room  temperature.  A  white  precipitate  is  formed. 
Shake  well  and  allow  to  stand  for  half  an  hour.  Filter  and  treat 
the  precipitate  with  water.  It  does  not  dissolve. 


D.    Colour  Reactions. 

21.  The  Xanthoproteic  reaction.     To    3  cc.  of    the   protein 
solution  in  a  test-tube  add  about  one  cc.  of  strong  nitric  acid.     A 
white  precipitate  is  formed  (see  Ex.  40).     Boil  for  a  minute.     The 
precipitate  turns  yellow  and  partly  dissolves  to  give  a  yellow  solu- 
tion.    Cool  under  the  tap  and  add  strong  ammonia  or  soda  till  the 
reaction  is  alkaline.    The  yellow  colour  is  turned  to  orange. 

NOTES. — i.  The  essential  features  of  the  reaction  are  that  a  yellow  colour 
is  obtained  when  the  solution  is  boiled  with  strong  nitric  acid,  and  that  this 
yellow  colour  is  intensified  when  the  solution  is  made  alkaline. 

2.  The  precipitate  is  due  to  the  formation  of  metaprotein  by  the  action 
of  nitric  acid  on  albumins  or  globulins,  this  metaprotein  being  insoluble  in 
strong  mineral  acids.     It  follows  that  proteoses  and  peptones,  etc.,  do  not  give 
the  precipitate  with  nitric  acid. 

3.  The  yellow  colour  is  due  to  the  formation  of  a  nitro-compound  of  some 
aromatic  substance,  i.e.  a  substance  containing  the  benzene  ring. 

4.  The  aromatic  substances  in  the  protein  molecule  that  are  responsible 
for  the  reaction  are  tyrosine,  tryptophane  and  phenyl  alanine. 

5.  Oleic  acid,  olive  oil  and  most  vegetable  oils  give  a  well-marked  xantho- 
proteic  reaction. 

6.  To  test  for  traces  of  proteins  proceed  as  follows  :   Boil  with  nitric  acid 
and  divide  into  two  portions.     Cool  one  portion  and  make  it  alkaline  with 
ammonia.     Compare  the  colour  of  the  two  portions.     The  alkaline  tube  will 
shew  a  faint  yellow  colour  when  only  the  merest  trace  of  protein  is  present. 

22.  Millon's   reaction.     Treat  5  cc.  of  the  protein  solution 
with  half  its  volume  of  Millon's  reagent.     A  white  precipkate  is 
formed.      Cautiously  heat   the  mixture.      The  precipitate  turns 
brick-red  in  colour,  or  disappears  and  leaves  a  red  solution. 

NOTES. — i.  The  essential  feature  of  the  reaction  is  the  red  colour  on 
heating.  The  white  precipitate  in  the  cold  is  due  to  the  action  of  the  mercuric 
nitrate  on  the  proteins.  (See  Ex.  9.) 

2.  A  white  precipitate  is  also  obtained  with  solutions  of  urea.     (See 
Exs.  341  and  342.) 

3.  Sulphates  give  a  white  precipitate  of  mercurous  sulphate. 


II.] 


COLOUR   TESTS. 


39 


4.  The  reagent  is  prepared  by  dissolving  one  part  by  weight  of  mercury 
in  twice  its  weight  of  concentrated  nitric  acid  (Sp.  gr.  i'42).     The  mixture  is 
slightly  warmed  towards  the  end.     It  is  then  treated  with  twice  its  bulk  of 
distilled  water,  allowed  to  settle  over-night,  and  filtered.   It  contains  mercurous 
and  mercuric  nitrates,  excess  of  nitric  acid,  and  a  small  amount  of  nitrous  acid. 

5.  The  reaction  should  never  be  attempted  with  a  strongly  alkaline  fluid, 
since  the  alkali  will  precipitate  the  yellow  or  black  oxides  of  mercury. 

6.  If  an  excess  of  the  reagent  be  employed  the  red  colour  is  often  dis- 
charged on  boiling. 

7.  The  reaction  is  given  with  all  aromatic  substances  that  contain  a 
hydroxyl  group  attached  to  the  benzene  ring.     Thus  it  is  given  by  phenol, 
salicylic  acid,  and  naphthol,  but  is  not  given  by  benzoic  acid. 


The  aromatic  substance  derived  from  protein  that  is  responsible  for  the 

"OH    (i) 

.CH2.CH.NH2.COOH     (4)' 


8. 


reaction  is  tyrosine.      C6H4= 


(See  Ex.  92.) 


23.  The  glyoxylic  reaction.  (Hopkins  and  Cole.)  Treat  2 
or  3  cc.  of  the  fluid  with  the  same  bulk  of  "  reduced  oxalic  acid  " 
("glyoxylic  reagent  ").  Mix  and  add  an  equal  volume  of  con- 
centrated sulphuric  acid,  pouring  it  down  the  side  of  the  tube. 
A  purple  ring  forms  at  the  junction  of  the  fluids.  Mix  the  fluids 
by  shaking  the  tubes  gently  from  side*  to  side.  The  purple  colour 
spreads  through  the  whole  fluid. 

NOTES. — i.  The  "glyoxylic  reagent "  is  prepared  by  one  of  the  follow- 
ing methods  : — 

A.  Treat  half  a  litre  of  saturated  solution  of  oxalic  acid  with  40 
grammes  of  2  per  cent,  sodium  amalgam  in  a  tall  cylinder.     When  all  the 
hydrogen  has  been  evolved  the  solution  is  filtered  and  diluted  with  twice 
its  volume  of  distilled  water.     The  solution  now  contains  oxalic  acid, 
sodium  binoxalate,  and  glyoxylic  acid  (COOH.CHO).     It  should  be  kept 
in  a  closed  bottle  containing  a  little  chloroform. 

B.  In  a  flask  place  10  grammes  of  powdered  magnesium  and  just 
cover  with  distilled  water.     Slowly  add  250  cc.  of  saturated  oxalic  acid, 
cooling  under  the  tap  at  intervals.     Filter  off  the  insoluble  magnesium 
oxalate,  acidify  with  acetic  acid,  dilute  to  one  litre  with  distilled  water,  and 
bottle  as  above. 

2.  The  reaction  does  not  succeed  in  the  presence  of  nitrates,  chlorates, 
nitrites,  or  excess  of  chlorides. 

3.  The  colour  is  not  well  seen  if  the  protein  is  mixed  with  certain  carbo- 
hydrates (e.g.  cane-sugar),  owing  to  the  char  produced  by  the  strong  sulphuric 
acid. 

4.  It  is  important  to  use  pure  sulphuric  acid  for  this  test.     It  sometimes 
fails  owing  to  the  presence  of  impurities  in  the  acid  employed.    At  the  same 
time  it  must  be  admitted  that  a  very  minute  trace  of  ferric  chloride  does 
sometimes  increase  the  intensity  of  the  colour. 


40  THE    PROTEINS.  [CH.    II. 

5.  In  performing  the  test  on  a  solid  substance,  like  fibrin,  or  keratin,  a 
small  amount  of  the  material  should  be  heated  with  a  few  cc.  of  the  reduced 
oxalic  acid  and  an  equal  volume  of  strong  sulphuric  acid.     The  mixture  is 
shaken,  and  as  the  protein  dissolves  in  the  strong  acid  both  the  fluid  and  the 
solid  particles  assume  a  purple  colour. 

6.  The  substance  in  the  protein  molecule  that  is  responsible  for  the 
reaction  is  tryptophane  (indol-amino-propionic-acid)  C11H12N2O2. 


NH2 
C   CH8.CH   COOH 


7.  A  similar  reaction  can  be  obtained  by  using  a  very  dilute  (1:250) 
solution  of  formaldehyde  containing  a  trace  of  an  oxidising  reagent  like  ferric 
chloride.     The  authors  of  the  original  reaction  regarded  this  test  (Rosenheim's) 
as  being  different  from  the  glyoxylic  test,  though  the  whole  question  is  still 
confused. 

8.  The  author  has  shown  that  many  substances,  especially  aldehydes, 
react  with  tryptophane  to  yield  coloured  products  in  the  presence  of  strong 
HC1  or  H2SO4.      Most  of  these  reactions  only  succeed  in  the  presence  of  an 
oxidising  reagent,  and  are  possibly  due  to  a  reaction  with  some  oxidation  pro- 

of  tryptophane. 


24.  The  biuret  reaction  (Piotrowski's  reaction).  Treat  about  3 
cc.  of  the  solution  with  I  cc.  of  40  per  cent,  sodium  hydroxide.  Add 
a  single  drop  of  a  I  per  cent,  solution  of  copper  sulphate.  A  violet 
or  pink  colour  is  produced. 

NOTES.  —  i  .  The  reaction  is  of  especial  importance  in  testing  for  proteoses 
and  peptones,  which  give  a  rose  colour.  It  is  generally  stated  that  other  pro- 
teins give  a  violet  colour,  but  usually  egg-albumin  gives  a  distinct  rose  tint. 

2.  It  is  important  to  avoid  an  excess  of  copper  sulphate,  the  blue  copper 
colour  obscuring  the  violet  or  rose  tint. 

3.  The  test  cannot  be  applied  in  the  presence  of  a  large  amount  of 
magnesium  sulphate,  owing  to  the  precipitation  of  magnesium  hydroxide  by 
the  alkali. 

4.  If  the  solution  contains  much  ammonium  sulphate  it  must  be  treated 
with  a  large  excess  of  strong  sodium  hydroxide,  as  directed  in  Ex.  57. 

5.  The  reaction  is  given  by  nearly  all  substances  containing  two 

O    H 
II      I 


CH.    II.] 


COLOUR   TESTS. 


41 


groups  attached  to  one  another,  to  the  same  nitrogen  atom,  or  to  the  same 
carbon  atom.  Thus  it  is  given  by 

CONK,,  CONHa  CONH2 

CONHa  NH  CH2 

CONH2  CONH2  ' 

Oxamide.  Biuret  (See  Ex.  345).         Malonamide. 

The  cause  of  the  reaction  with  proteins  is  the  presence  of  one  or  more  groupings 
of  the  last  type,  formed  by  the  condensation  of  the  carboxylic  group  of  an 
amino-acid  with  the  amino  group  of  another  amino-acid.  The  linkage  thus 


formed  is  known  as  the  "  pep  tide  "  linkage, 
polypeptide,  glycyl-alanyl-tyrosine 


Thus  it  would  be  given  by  the 


! 

CH3 

NH2.CH2.CO.  NH.CH.CO. 
Glycyl-j       -alanyl- 

CflH4.OH 
CH2 

NH.CH.COOH. 

-tyrosine. 

25.  The  sulphur  reaction.  Boil  a  little  undiluted  egg-white 
or  serum  with  some  40  per  cent,  sodium  hydroxide  for  two  minutes, 
and  then  add  a  drop  or  two  of  lead  acetate.  The  solution  turns  deep 
black. 

NOTES. — i .  This  reaction  is  due  to  the  fact  that  the  sulphur  of  the  protein 
is  liberated  as  sodium  sulphide  when  boiled  with  the  strong  alkali.  The 
sulphide  gives  a  black  colour  or  precipitate  of  lead  sulphide  when  the  solution 
is  subsequently  treated  with  lead  acetate. 

2.  The  reaction  does  not  succeed  with  caseinogen,  peptones,  and  certain 
other  proteins. 

3.  The  sulphur  in  the  protein  is  mainly  combined  as 


CH2.SH 
CH.NH2 

COOH 
Cystein 


or 


CH2.S.S.CH2 

CH.NH2  CH.NH2 

I  I 

COOH      COOH 
Cystine 


26.  Molisch's  reaction.  Treat  5  cc.  of  the  diluted  solution 
with  three  or  four  drops  of  a  I  per  cent,  solution  of  alpha-naphthol  or 
of  thymol  in  alcohol.  Mix,  and  run  about  5  cc.  of  concentrated 
sulphuric  acid  under  the  fluid.  A  violet  ring  is  formed  at  the 
junction  of  the  two  liquids. 

NOTES. — i.  The  reaction  is  due  to  the  presence  of  a  carbohydrate  group 
(glucosamine)  in  the  protein.  This  is  converted  by  the  acid  to  furfurol,  which 
condenses  with  the  alpha-naphthol  or  the  thymol  to  give  the  purple  colour. 
(See  notes  to  Exs.  no  and  114.) 

2.  A  green  ring  is  often  seen  in  addition  to  the  violet  ring.  This  is  due 
to  the  action  of  the  sulphuric  acid  on  the  alpha-naphthol. 


42  THE    PROTEINS.  [cH.    II. 

E.    The  heat  coagulation  of  albumins  and  globulins. 

These  proteins  are  placed  in  a  class  by  themselves, 
because  they  exhibit  most  characteristically  the  phenome- 
non of  heat  coagulation. 

A  proper  understanding  of  the  conditions  governing 
this  phenomenon  is  so  important  that  students  are  urged 
to  study  them  attentively.  The  following  matter  should 
be  re-read  after  the  section  on  metaproteins  has  been 
studied. 

When  a  solution  of  albumin  or  globulin  is  heated  under 
certain  conditions  the  protein  separates  in  a  form  which  is 
insoluble  in  water,  dilute  salt  solutions,  acids  and  alkalies. 
This  is  the  phenomenon  known  as  "  heat  coagulation." 
The  term  "  coagulation  "  is  used  as  an  indication  of  an 
irreversible  change  and  to  distinguish  the  condition  of  the 
protein  from  that  of  "  precipitation/'  in  which  re-solution 
can  be  brought  about  by  a  change  of  reaction,  salt  content, 
etc. 

The  two  most  important  conditions  affecting  heat 
coagulation  are  reaction  and  salt  content.  It  will  be  found 
later  that  albumins  and  globulins  are  readily  converted  into 
metaproteins  by  treatment  with  acids  or  alkalies,  the  con- 
version being  much  accelerated  by  a  rise  in  temperature. 
The  metaproteins  are  soluble  in  dilute  acids  or  alkalies,  but 
are  insoluble  in  the  neutral  condition,  i.e.  in  water  or 
neutral  salt  solutions.  An  important  fact  about  them  is 
that  if  a  precipitate  of  metaprotein  is  boiled  it  is  "  coagu- 
lated," that  is,  it  will  not  redissolve  in  dilute  acids  or 
alkalies.  The  conversion  of  albumin  or  globulin  into  meta- 
protein is  called  "  denaturation,"  and  is  a  necessary  ante- 
cedent of  heat  coagulation.  This  process  is  best  regarded 
as  an  hydrolysis,  which  takes  place  at  all  reactions,  but 
most  rapidly  in  either  acid  or  alkaline  solutions.  At  boil- 
ing point  the  change  is  practically  instantaneous,  no  matter 
what  the  reaction  may  be.  Should  the  reaction  be  at  the 
iso-electric  point  of  the  denaturised  protein  either  the  whole  or 


CH.   II.]  HEAT   COAGULATION.  43 

a  considerable  part  of  this  is  aggregated  into  flocks  (see 
p.  11).  At  high  temperatures  these  flocks  are  coagulated, 
that  is,  they  do  not  redissolve  on  altering  the  reaction  of  the 
fluid. 

The  main  effect  of  neutral  salts  is  to  aid  the  aggrega- 
tion of  the  denaturised  protein.  It  often  happens  that 
more  than  one  denaturised  protein  is  formed  by  heating  a 
solution  containing  albumins  and  globulins.  The  iso- 
electric  points  of  these  proteins  may  differ,  so  that  a  certain 
proportion  of  the  protein  will  remain  non-coagulated  at  any 
given  reaction.  This  seems  to  be  true,  even  in  the  case  of 
the  denaturised  protein  formed  from  a  pure  protein.  The 
addition  of  a  neutral  salt  will  tend  to  cause  flocking  of  this 
"  soluble  "  portion  of  the  denaturised  protein,  and  these 
flocks  will  be  coagulated  should  the  temperature  be  high 
enough.  The  non-coagulated  dispersed  protein  carries  an 
electric  charge,  which  varies  with  the  reaction,  being  posi- 
tive in  acid  solutions  and  negative  in  alkaline  solutions. 
We  have  seen  that  electro-positive  colloids  are  flocked  by 
negative  ions,  and  electro-negative  colloids  by  positive 
ions.  Further,  that  this  flocking  power  of  the  ions  is  much 
greater  with  di-  and  tri-valent  ions  than  with  mono-valent 
ions.  It  follows,  therefore,  that  the  ideal  conditions  for  the 
maximal  heat  coagulation  of  an  albumin  or  globulin  are 
that  the  reaction  should  be  at  the  iso-electric  point  of  the 
denaturised  protein  formed,  and  that  there  should  be 
present  di-  or  tri-valent  ions  both  positive  and  negative. 
These  conditions  are  met  by  having  the  boiling  solution 
very  faintly  acid  to  litmus  and  adding  a  trace  of  calcium 
chloride  or  magnesium  sulphate.  It  is  advisable  to  add  the 
salt,  to  boil  the  solution,  and  then  to  change  the  reaction 
slowly  by  the  addition  of  dilute  sodium  carbonate  or  i 
per  cent,  acetic  acid,  so  that  the  final  reaction  is  just  acid 
to  litmus.  The  exact  point  and  procedure  can  only  be 
determined  by  experience  since  it  varies  considerably  with 
the  concentration  of  the  protein,  etc. 


44  THE    PROTEINS.  [CH.    II. 

For  the  following  five  exercises  use  serum  that  has  been  diluted  with  10 
volumes  of  distilled  water. 

27.  Boil  5  cc.  in  a  test-tube  that  has  been  previously  rinsed 
with  distilled  water.     The  solution  becomes  opalescent,  but  usually 
no  definite  coagulum  is  formed.     Cool  the  tube  and  add  i  per  cent, 
acetic  acid  drop  by  drop.     A  precipitate  of  metaprotein  is  formed 
soluble  in  excess  of  acid. 

NOTE.— The  reaction  of  the  mixture  after  boiling  is  distinctly  alkaline  to 
the  iso-electric  point  of  the  denaturised  proteins  formed.  These  are  precipi- 
tated by  bringing  the  reaction  of  the  solution  to  the  iso-electric  point  by  the 
addition  of  acetic  acid,  but  are  redissolved  by  an  excess. 

28.  To  5  cc.  add  two  drops  of  i  per  cent,  acetic  acid  and  boil. 
A  white  flocculent  coagulum  is  formed.     Cool  the  tube  and  add  two 
or  three  drops  of  strong  nitric  or  acetic  acid.     The  coagulum  does 
not  dissolve. 

NOTES. — i.  The  amount  of  acid  added  is  such  that,  after  boiling,  the 
reaction  is  near  to  the  iso-electric  point  of  the  denaturised  proteins.  These 
are  therefore  precipitated  and  then  coagulated. 

2.  The  addition  of  the  strong  acid  is  to  ensure  that  the  precipitate  that 
appears  on  boiling  does  not  consist  of  calcium  or  magnesium  phosphate,  which 
is  soluble  in  dilute  nitric  acid.  That  such  a  phosphatic  precipitate  can  be 
formed  on  boiling  certain  solutions  is  shown  by  the  following  experiment. 
Treat  a  solution  of  calcium  chloride  with  sodium  phosphate  and  then  with 
excess  of  sodium  carbonate.  A  precipitate  of  Ca3(PO4).j  appears.  Add  acetic 
acid  drop  by  drop  till  the  precipitate  just  dissolves  owing  to  the  formation  of 
the  acid  phosphate.  Boil  the  solution  for  half  a  minute.  A  white  precipitate 
appears.  Add  a  drop  or  two  of  nitric  acid.  The  precipitate  dissolves.  The 
appearance  of  the  precipitate  of  Ca3(PO4)2  on  boiling  is  due  to  the  alteration  of 
reaction  as  the  COa  is  evolved. 

29.  Treat  5  cc.  of  the  solution  with  0-4  per  cent,  hydrochloric 
acid,  drop  by  drop,  until  the  precipitate  obtained  by  the  first  drop 
or  two  has  redissolved  (about  five  drops  are  usually  necessary). 
Boil.     The  solution  remains  clear.     Cool  the  tube  and  add  2  per  cent, 
sodium  carbonate,  drop  by  drop.     A  precipitate  of  metaprotein  is 
formed  which  redissolves  in  excess. 

NOTE. — The  precipitate  that  first  forms  consists  of  globulin  (see  Ex.  32). 
On  adding  enough  HCl  to  redissolve  this  the  reaction  is  such  that  it  is  acid  to  the 
iso-electric  point  of  the  denaturised  proteins.  These  are  precipitated  by  an 
alkali  and  redissolve  in  an  excess. 

30.  To  5  cc.  add  two  drops  of  2  per  cent,  sodium  carbonate 
and  boil.     The  solution  remains  quite  clear.     Cool  the  tube  and  add 
i  per  cent,  acetic  acid,  drop  by  drop.     A  precipitate  of  metaprotein 
is  formed,  soluble  in  excess. 


CH.    II.]  ALBUMINS   AND   GLOBULINS.  45 

NOTE. — The  results  in  this  exercise  are  similar  to  those  obtained  in  Ex.  27, 
except  that  the  increased  alkalinity  prevents  the  formation  of  the  opalescence 
obtained  in  the  absence  of  added  alkali. 

31.  To  5  cc.  add  a  drop  of  2  per  cent,  calcium  chloride  and  boil. 
A  considerable  coagulum  is  obtained. 

NOTE. — Though  the  solution  is  alkaline  to  the  iso-electric  point,  the  di- 
valent positive  calcium  ion  precipitates  a  certain  proportion  of  the  negative 
colloidal  protein. 

F.    The  properties  of  albumins  and  globulins. 

Globulins  are  generally  insoluble  in  distilled  water,  but 
soluble  in  dilute  acids  and  alkalies,  and  in  weak  solutions 
of  neutral  salts. 

A  neutral  solution  in  a  dilute  salt  is  coagulated  on 
boiling. 

A  solution  in  dilute  acid  or  alkali  is  converted  into  a 
solution  of  metaprotein  on  boiling. 

If  the  globulin  be  dissolved  in  a  minimum  amount  of 
a  neutral  salt  solution  and  the  solution  be  diluted  with 
several  volumes  of  distilled  water,  the  globulin  is  partially 
precipitated,  for  a  certain  concentration  of  salt  is  necessary 
to  keep  the  globulin  in  solution.  If  the  globulin  be  dis- 
solved in  dilute  acid  or  alkali,  there  is  no  precipitation  on 
dilution. 

The  globulins  are  completely  precipitated  by  full 
saturation  with  magnesium  sulphate  or  by  half-satura- 
tion with  ammonium  sulphate,  i.e.  by  treatment  of  the 
solution  with  an  equal  volume  of  a  saturated  solution  of 
ammonium  sulphate. 

Albumins  are  soluble  in  distilled  water,  dilute  salt 
solutions,  dilute  acids  and  alkalies. 

A  neutral  solution  in  water  or  salt  is  coagulated  on  boil- 
ing. 

A  solution  in  dilute  acid  or  alkali  is  converted  to  a 
solution  of  metaprotein  on  boiling. 

Solutions  of  albumins  are  not  precipitated  by  saturation 
with  magnesium  sulphate  nor  by  half-saturation  with 


46  THE   PROTEINS.  [CH.    II. 

ammonium  sulphate  if  the  reaction   of  the  solution  be 
neutral  or  alkaline. 

They  are  partially  precipitated  by  solutions  of  these 
substances  in  the  presence  of  acid. 

They  are  completely  precipitated  by  full  saturation 
with  ammonium  sulphate  from  a  neutral,  acid,  or  alkaline 
solution. 

The  solubility  in  water  of  the  globulins  of  blood  serum  is  apparently 
modified  by  the  presence  of  certain  "  lipines  "  (see  p.  153).  If  the  serum 
globulins  be  precipitated  by  half  saturation  with  ammonium  sulphate,  the  pre- 
cipitate dissolved  in  water,  as  described  in  Ex.  36,  and  the  solution  thoroughly 
dialysed,  it  will  be  found  that  only  a  portion  of  the  protein  is  precipitated  by 
the  dialysis.  The  fraction  that  remains  soluble  in  water  has  been  called 
"  pseudo-globulin  "  to  distinguish  it  from  the  water-insoluble  fraction  or 
"  eu-globulin."  It  was  formerly  believed  that  "  pseudo-globulin "  was 
an  albumin  and  that  it  was  impossible  to  separate  the  globulins  from  the 
albumins  by  half  saturation  with  ammonium  sulphate.  Recent  work  by 
Hartley  on  the  globulins  of  serum  and  by  Raistrick  on  those  of  milk  have 
demonstrated,  however,  that  the  distribution  of  nitrogen  as  mon-amino  acids 
and  as  bases  is  practically  the  same  for  the  two  globulin  fractions,  which  differ 
appreciably  from  the  albumin  fraction  in  this  respect.  It  is  probable  that 
the  insolubility  of  the  "  eu-globulin  "  in  water  is  due  to  its  association  with 
lipoid. 

32.  Dilute  5  cc.  of  serum  with  50  cc.  of  distilled  water.     A 
faint  cloud  of  serum  globulin  is  formed.     Cautiously  add  0-4  per 
cent,  hydrochloric  acid  or  i  per  cent,  acetic  acid  until  the  cloud  has 
reached  its  maximum  density.     Divide  into  two  portions  A  and  B. 
To  A  add  a  couple  of  drops  of  a  saturated  solution  of  ammonium 
sulphate.     The  solution  becomes  quite  clear.     To  B  add  a  couple  of 
drops  of  strong  acid.     The  cloud  disappears. 

NOTE. — The  globulin  of  the  serum  is  held  in  solution  both  by  salts  and 
alkalies.  Dilution  alone  produces  a  very  small  precipitate,  but  if  the  solution 
be  now  treated  with  just  sufficient  acid  to  neutralise  the  alkali,  a  much  larger 
fraction  of  the  globulin  is  thrown  down.  This  globulin  is  soluble  in  dilute 
neutral  salts,  or  in  an  excess  of  acid. 

33.  Prepare  a  suspension  of  globulin  by  the  following  method. 
To  15  cc.  of  serum  in  a  beaker  add  2  cc.  (about  30  drops)  of  I  per 
cent,  acetic  acid  and  100  cc.  distilled  water.     Stir  and  allow  the 
mixture  to  stand  for  about  20  minutes.     A  precipitate  of  globulin 
settles  down.    Very  carefully  pour  off  the  supernatant  fluid  and 
divide  the  suspended  globulin  into  two  equal  portions  in  clean  test- 
tubes.     With  these  perform  the  two  following  exercises. 


GLOBULINS. 


47 


34.  Add  a  5  per  cent,  solution  of  sodium  chloride,  drop  by  drop, 
till  the  globulin  has  jusT  dissolved.     Divide  the  solution  into  three 
portions,  (a),  (b)  and  (c). 

(a)  Boil.    The  protein  is  coagulated. 

(b)  Dilute  with  about  five  volumes  of  distilled  water.     The 

globulin  is  partially  reprecipitated. 

(c)  Treat  with  an  equal  volume  of  saturated  ammonium  sulphate 

solution.     The  globulin  is  reprecipitated. 

35.  Add  0-4  per  cent.  H  Cl,  drop  by  drop,  till  the  globulin  has 
just  dissolved.     Divide  the  solution  into  three  portions,  (d),  (e)  and 


(d)  Add  2  per  cent,  sodium  carbonate  solution  till  the  globulin 

is  partially  reprecipitated  (one  or  two  drops  only  are 
necessary).  Now  add  a  few  drops  of  5  per  cent,  sodium 
chloride.  The  precipitate  of  globulin  redissolves. 

(e)  Boil  the  solution.     The  protein  is  not  coagulated.     Cool 

under  the  tap  and  add  enough  2  per  cent,  sodium  carbon- 
ate to  precipitate  the  metaprotein  that  has  been  formed 
by  boiling.  Now  add  a  few  drops  of  5  per  cent,  sodium 
chloride.  The  precipitate  of  metaprotein  does  not 
dissolve. 


(/)  Dilute  with  about  five  volumes  of  distilled  water, 
globulin  is  not  thrown  out  of  solution. 


The 


36.  Mix  about  10  cc.  of  undiluted  serum  with  an  exactly  equal 
quantity  of  a  saturated  solution  of  ammonium  sulphate.    A  thick 
white  precipitate  is  formed  consisting  of  the  whole  of  the  globulin. 
Filter  through  a  dry  filter  paper  into  a  dry  test-tube.      Label  the 
filtrate  A.     Scrape  the  precipitate  off  the  paper  and  treat  it  with 
distilled  water.    The  precipitate  dissolves,  the  ammonium  sulphate 
adhering  to  it  forming  a  dilute  salt  solution  which  allows  the  globulin 
to  go  into  solution.      Boil  a  portion  of  this  solution.     A  heat- 
coagulum  is  formed. 

37.  Filtrate  A  contains  serum-albumin  in  the  presence  of 
half-saturated  ammonium  sulphate.    Apply  the  following  tests : 

(a}  Boil  a  portion.     A  heat-coagulum  is  formed, 


ot 


48  THE   PROTEINS.  [CH.   II. 

(b)  To  another  add  one  drop  of  strong  acetic  acid.    A  white 

precipitate  of  serum-albumin  i?  formed. 

(c)  Grind  the  remainder  in  a  mortar  with  solid  ammonium 

sulphate,  till  the  fluid  is  saturated.  A  white  precipitate 
of  serum-albumin  is  formed.  Filter  off  the  precipitate 
and  test  the  nitrate  for  proteins  either  by  boiling  or  by 
the  glyoxylic  or  xanthoproteic  reactions.  Proteins  are 
absent,  showing  that  all  the  proteins  of  serum  are  precipi- 
tated by  complete  saturation  with  ammonium  sulphate. 

NOTE. — A  certain  test  for  albumin  in  a  solution  is  to  half-saturate  it  with 
ammonium  sulphate,  filter  off  any  precipitate  that  may  be  present  and  boil  the 
filtrate.  A  heat-coagulum  indicates  albumin. 

38.  Serum  has  been  dialysed  in  collodion  sacs   (see  p.  2)  for 
24  hours  against  distilled  water  in  a  tall  cylinder.     Note  the  heavy 
precipitate  of  serum-globulin  that  has  fallen  to  the  bottom  of  the 
sac.     Pipette  off  some  of  the  clear  fluid  and  add  an  equal  volume  of 
saturated  ammonium  sulphate.    A  precipitate  of  "pseudo-globulin  " 
(see  note  on  p.  46)  is  obtained.     Now  pipette  off  some  of  the  deposit, 
add  about  two  volumes  of  distilled  water,  and  divide  into  three 
portions,  A,  B,  and  C.     To  A  add  a  couple  of  drops  of  saturated 
ammonium  sulphate.     To  B  add  a  drop  of  dilute  soda.    To  C  add  a 
drop  or  two  of  dilute  HC1.     The  globulin  dissolves  in  each  case. 

39.  Dilute  3  cc.  of  serum  with  about  five  times  its  volume  of 
tap  water,  add  a  drop  of  2  per  cent,  calcium  chloride,  and  boil  the 
mixture  in  a  boiling  tube.     Add  one  drop  of  I  per  cent,  acetic 
acid  and   boil   again.     Continue  this  procedure  until  a  definite 
coagulum  has  formed,  and  the  fluid  between  the  flocks  appears  to  be 
clear  when  examined  in  a  thin  layer.     Filter.     The  filtrate  should 
run  through  the  paper  rapidly  and  be  crystal  clear.     If  it  filters 
slowly  or  comes  through  opalescent,  repeat  the  experiment  until 
the  desired  result  is  obtained.     Test  the  filtrate  for  proteins  by 
Millon's  and  the  xanthoproteic  tests.     Only  insignificant  traces 
should  be  found. 

NOTE. — This  is  the  method  usually  adopted  for  removing  albumins  and 
globulins  from  solution,  but  it  must  be  noted  that  it  is  almost  impossible  to 
remove  the  last  traces  by  this  procedure.  If  it  is  necessary  to  do  so,  colloidal 
iron  (see  Ex.  310),  metaphosphoric  acid  (see  Ex.  17),  or  some  other  reagent  must 
be  employed.  The  objection  to  the  use  of  such  reagents  is  that  they  are  apt 
to  precipitate  the  proteoses,  peptones,  etc. 


CH.    II.]  EGG-WHITE.  49 

40.  The  action  of  mineral  acids  on  albumins  and  globulins. 
(Heller's  test.)  Place  a  few  cc.  of  strong  nitric  acid  in  a  narrow 
test-tube.  By  means  of  a  pipette  add  an  equal  volume  of  dilute 
serum  or  egg-white,  inclining  the  tube  during  the  addition  so  that 
the  protein  solution  is  "layered"  on  the  surface  of  the  acid.  A 
white  ring  appears  at  or  immediately  above  the  junction  of  the 
two  fluids. 

NOTE. — This  is  one  of  the  most  important  tests  for  albumins  in  urine. 
The  reaction  is  also  given  by  HC1  and  H2SO4,  but  not  so  readily  as  by  HNO3. 
The  primary  proteoses  also  give  a  precipitate  but  this  is  soluble  on  warming. 


G.    The  chemistry  of  egg-white. 

41.  In  egg-white  which  has  been  well  beaten  with  a  whisk 
(to  break  up  the  containing  membranes),  and  diluted  with  four  times 
its  volume  of  distilled  water,  note  a  precipitate  of  ovo-mucin  and 
globulin.  Perform  the  following  tests : 

(a)  Take  the  reaction  to  litmus.     It  is  alkaline. 

(b)  Cautiously  neutralise   with   dilute   acetic   acid.     A   slight 

increase  in  the  precipitate  of  ovo-mucin  and  globulin  is 
noticed.  Remove  this  by  nitration  if  necessary,  and 
with  the  nitrate  perform  the  following  reactions : 

(c)  Boil   a  portion.     A   coagulum  is   formed,   indicating   the. 

presence  of  either  a  globulin  or  an  albumin. 

(d)  Make  another  portion  very  faintly  alkaline  by  the  addition 

of  a  drop  or  two  of  2  per  cent.  Na2CO3.  Now  add  an 
equal  bulk  of  saturated  (NH4)2SO4.  A  slight  precipitate 
of  globulin  or  albumin  is  formed.  Filter  this  off,  and 
boil  a  portion  of  the  nitrate  with  a  drop  of  I  per  cent, 
acetic  acid.  A  coagulum  of  albumin  is  formed.  Saturate 
the  remainder  of  this  nitrate  with  ammonium  sulphate 
by  grinding  with  the  solid  in  a  mortar.  A  precipitate 
of  albumin  is  formed. 

(e)  Completely  remove  the  globulin  and  albumin  by  boiling. 

Filter  and  apply  Millon's  or  the  xanthoproteic  protein 
test  to  the  filtrate.  Protein  is  found  in  small  quantities. 


50  THE   PROTEINS.  [CH.    II. 

This  protein  is  known  as  ovo-mucoid.  It  is  not  coagu- 
lated by  boiling,  nor  precipitated  by  acetic  acid.  It  is 
precipitated  by  saturation  with  ammonium  sulphate,  and 
also  by  strong  alcohol. 

42.  The  crystallisation  of  egg-albumin.  (Hopkins'  method.) 
Separate  the  white  from  a  number  of  new-laid  eggs,  taking  care  not  to 
allow  any  of  the  yolk  to  mix  with  the  white.  Measure  the  egg-white 
and  churn  it  up  with  an  exactly  equal  volume  of  a  neutral  fully- 
saturated  solution  of  ammonium  sulphate  by  means  of  a  whisk, 
adding  the  sulphate  in  portions  and  mixing  thoroughly  after  every 
addition.  Notice  the  strong  smell  of  ammonia  that  is  evolved. 
Filter  the  mixture  through  a  large  pleated  filter-paper.  Measure 
the  filtrate.  Take  100  cc.  of  it  and  cautiously  treat  it  with  10  per 
cent,  acetic  acid  from  a  burette,  noting  the  original  level  of  the  acid 
in  the  burette.  Add  the  acid  a  drop  or  two  at  a  time,  shaking  gently 
the  whole  time,  until  the  precipitate  produced  at  each  addition  no 
longer  dissolves  on  shaking,  and  the  whole  mixture  is  rather  opale- 
scent. This  point  is  usually  somewhat  difficult  to  determine,  owing 
to  the  large  number  of  air-bubbles  that  become  suspended  in  the 
fluid  and  closely  resemble  a  fine  precipitate.  When  you  are  satisfied 
that  a  permanent  precipitate  has  been  produced,  run  in  i  cc.  of  the 
acid  in  addition  to  the  amount  already  added.  A  heavy  white  precipi- 
tate is  thus  produced.  Note  the  amount  of  acid  that  has  been  used 
for  the  portion  of  100  cc.,  and  treat  the  remainder  of  the  filtrate  with 
a  corresponding  amount  of  acid.  Mix  the  two  portions  thoroughly 
and  allow  to  stand  overnight.  Note  that  the  precipitate  has  in- 
creased somewhat  in  amount.  Mount  a  drop  of  the  suspension  on  a 
slide,  cover  with  a  slip,  but  do  not  press.  Examine  under  the  high 
power  of  the  microscope,  and  note  the  aggregation  of  very  fine 
needles. 

The  albumin  can  be  recrystallised  by  filtering,  dissolving  in  as 
small  an  amount  of  water  as  possible,  filtering  again,  and  cautiously 
adding  to  the  filtrate  saturated  ammonium  sulphate  till  a  faint 
permanent  precipitate  is  produced.  If  the  mixture  be  allowed  to 
stand  for  some  hours  the  albumin  will  separate  out  as  fine  needles. 

NOTES. — i.  For  the  experiment  to  succeed  it  is  absolutely  essential  that 
all  theTeggs  employed  be  perfectly  fresh.  One  rather  stale  egg  may  interfere 
with  ^the  crystallisation  of  a  large  number  of  fresh  eggs. 


METAPROTEINS. 


51 


2.  It  is  important  to  add  exactly  the  amount  of  acetic  acid  mentioned, 
that  is,  one  per  mille  above  the  amount  required  to  give  a  faint  permanent 
precipitate. 

3.  The  same  method  can  be  employed  for  the  crystallisation  of  serum- 
albumin  from  the  perfectly  fresh  serum  of  a  horse,  ass  or  mule. 


H.    The  Metaproteins. 

The  metaproteins  are  derived  from  the  albumins  and 
globulins  by  hydrolysis.  This  can  be  effected  rapidly  by 
dilute  acids  and  alkalies  at  temperatures  over  60°  C.  (see 
Exs.  29  and  30) :  more  slowly  at  body  temperature.  They  are 
formed  immediately  by  the  action  of  strong  mineral  acids 
at  room  temperature.  They  are  insoluble  in  water,  strong 
mineral  acids,  and  all  solutions  of  neutral  salts,  but  are 
soluble  in  dilute  acids  or  alkalies  in  the  absence  of  any 
large  amount  of  neutral  salts.  They  are  not  thrown  out 
of  solution  (in  acid  or  alkali)  by  boiling.  But  if  such 
a  solution  be  neutralised  or  precipitated  by  the  addition  of 
an  excess  of  a  neutral  salt,  the  suspended  metaprotein  is 
coagulated  on  boiling,  so  that  it  will  no  longer  dissolve  in 
acid  or  alkali. 

Preparation.  Egg  white  or  serum  is  diluted  with  ten  times  its  volume 
of  either  0-4  per  cent,  hydrochloric  acid  or  o-i  per  cent,  sodium  hydroxide  and 
the  mixture  placed  in  a  water  bath  or  incubator  at  40°  C.  for  about  twenty-four 
hours.  The  albumins  and  globulins  are  hydrolysed  to  metaprotein. 

43.  Neutralise  about  25  cc.  with  2  per  cent,  sodium  carbonate, 
or  0-4  per  cent.  HC1,  depending  on  the  original  reaction  of   the 
fluid.     A  bulky  precipitate  of  metaprotein  forms.    The  acid  or 
alkali  should  be  added  until  the  maximum  amount  of  precipitate  is 
produced.     The  reaction  then  will  probably  be  very  slightly  acid 
to   litmus.     Filter.    The   nitrate   generally   comes   through   very 
slowly.     When  as  much  as  possible  of  the  fluid  has  been  removed  in 
this  way  transfer  the  fluid  on  the  paper  to  a  small  beaker,  open  the 
paper,  and  add  the  precipitate  to  the  fluid  that  has  been  poured  off, 
dilute  with  a  little  water,  and  divide  the  suspension  into  six  equal 
portions  and  with  them  perform  the  following  six  exercises. 

44.  Add  some  0-4  per  cent.  HC1.     The  precipitate  dissolves. 
Neutralise  with  sodium  carbonate:  the  precipitate  reappears. 


52  THE   PROTEINS.  [CH.    II. 

45.  Add  concentrated  HC1  drop  by  drop.      The  precipitate 
dissolves  with  the  first  drop,  but  generally  reappears  when  an  excess 
is  added  (see  Ex.  21,  note  2,  and  Ex.  40). 

46.  Dissolve  in  a  little  0-4  per  cent.  HC1.      Boil  the  solution  : 
a  coagulum  is  not  formed.     Cool  under  the  tap  and  neutralise  with 
0-2  per  cent.  Na2CO3.     A  precipitate  is  formed  which  is  soluble  in  an 
excess  of  the  alkali. 

47.  Boil.     Cool,    and   add   some   0-4   per   cent.    HC1.      The 
precipitate  does  not  dissolve,  i.e.  metaprotein  is  coagulated  when 
boiled  in  suspension. 

48.  Add  a  saturated  solution  of  ammonium  sulphate  drop  by 
drop.    The  precipitate  does  not  dissolve  in  any  dilution  of  the  salt. 
The  insolubility  in  dilute  solutions  of  neutral  salts  is  an  important 
distinction  between  metaproteins  and  globulins  (see  Ex.  32  and  34). 

49.  Dissolve  in  a  little  0-4  per  cent.  HC1.      Treat  the  solution 
with  an  equal  volume  of  saturated  ammonium  sulphate  solution. 
The  protein  is  precipitated. 


I.   The  Albumoses  or  Proteoses  and  Peptones. 

These  hydrolysed  proteins  are  obtained  by  the  further 
action  of  acids  or  alkalies  on  globulins,  albumins  and  meta- 
proteins. They  are  best  formed  by  the  action  of  pepsin  and 
hydrochloric  acid  on  these  proteins.  Peptone  is  the  end 
product  of  gastric  digestion. 

They  are  prepared  on  a  commercial  scale  and  sold  as— 

(i.)  Witte's  peptone,  which  is  prepared  from  fibrin 
and  consists  of  a  mixture  of  albumoses  and 
peptone. 

(ii.)  Savory  and  Moore's  peptone,  which  is  prepared 
from  meat,  and  only  contains  traces  of 
albumoses. 

The  following  scheme  indicates  the  successive  steps 


CH.    II.] 


PROTEOSES   AND    PEPTONES. 


53 


in  the   digestion  of  fibrin  by  pepsin  and  0-2   per  cent, 
hydrochloric   acid  : — 


Fibrin 

Soluble  Globulin 

I 
Metaprotein 


Primary  albumoses 

Proto-albumose  :  Hetero-albumose. 


Secondary  albumoses 

Thio-albumose  :  Synalbumose,  etc. 


Peptones. 

The  following  scheme  shews  the  method  adopted  for 
the  isolation  of  certain  of  the  albumoses  : — 

Neutral  Witte's  peptone,  treated   with  equal  volume   of 
saturated  ammonium  sulphate  solution. 


Precipitate  :    dis- 
solved in  water. 
Treated  with  2 
volumes  of  strong 
alcohol. 

Filtrate,  treated  with  half  its  volume  of  saturated 
ammonium  sulphate. 

Ppt.  dissolved 
in  water. 
Treated    with 
2  volumes  of 
alcohol. 

Ppt. 
Thio- 
albumose. 

Filtrate.      Saturated  with  ammo- 
nium sulphate. 

Ppt. 

Hetero- 
albumose. 

Filtrate. 

Proto- 
albumose. 

Ppt.  dissolved  in  water. 
Treated  with  2  volumes 
of  strong  alcohol. 

Filtrate. 
Peptones 

Ppt. 
Neglect. 

Filtrate. 

Treated  with 
f  vols.  of  alco- 
hol. 

Ppt. 
Synalbumose. 

The  primary  albumoses  are  soluble  in  water,  dilute 
acids,  alkalies  and  salt  solutions.  Their  solutions  are  not 
coagulated  on  heating.  They  are  precipitated  by  half- 
saturation  with  ammonium  sulphate.  They  give  a  pre- 


54  THE    PROTEINS.  [CH.    II. 

cipitate,  that  disappears  on  warming  and  reappears  on 
cooling,  either  with  nitric  acid  or  potassium  ferrocyanide 
and  acetic  acid.  They  also  give  a  precipitate  in  the  cold 
with  copper  sulphate. 

They  give  all  the  ordinary  protein  colour  reactions, 
with  the  exception  of  Molisch's. 

The  secondary  albumoses  have  somewhat  similar  pro- 
perties to  those  of  the  primary  albumoses  :  but  they  are 
not  precipitated  by  nitric  acid,  ferrocyanic  acid,  or  copper 
sulphate.  * 

They  require  more  than  half-saturation  with  ammo- 
nium sulphate  to  precipitate  them,  but  are  completely 
precipitated  by  full  saturation.  Thio-albumose  gives  all 
the  protein  colour  reactions  and  is  particularly  rich  in 
sulphur  (hence  its  name). 

Synalbumose  gives  the  protein  reactions,  with  the 
exception  of  the  glyoxylic  test. 

The  peptones  are  very  soluble  proteins  of  rather  a  low 
molecular  weight,  so  that  they  slowly  diffuse  through 
parchment  membranes.  They  are  the  only  proteins  not 
precipitated  by  full  saturation  with  ammonium  sulphate. 
They  fail  to  give  precipitates  with  Esbach's  and  Brucke's 
reagents  or  ferrocyanic  acid,  but  are  precipitated  by  other 
protein  precipitants,  as  tannic  acid,  phosphotungstic  acid 
and  lead  acetate. 

For  the  following  reactions  make  a  5  per  cent,  solution  of  "  Witte's 
peptone  "  in  hot  water,  just  acidify  with  acetic  acid  and  filter  from  a  small 
amount  of  insoluble  material  (nuclein?).  The  solution  contains  all  the 
albumoses  and  peptones. 

50.  Dilute  a  small  amount  with  three  or  four  times  its  bulk 
of  water,  and  to  portions  of  this  apply  the  usual  colour  reactions  for 
protein.     They  are  all  obtained.     Note,  in  particular,  that  the  biuret 
test  gives  a  rose  colour. 

51.  Boil  the  solution  with  a  trace  of  acetic  acid :  it  does  not 
form  a  coagulum. 


CH.    II.]  PROTEOSES   AND   PEPTONES.  55 

52.  Add  a  little  tannic  acid :  a  white  precipitate  is  formed. 

53.  Add  a  little  Esbach's  or  Briicke's  solution :  a  yellow  or 
white  precipitate  is  formed. 

54.  Add  a  little  lead  acetate  solution :  a  white  precipitate  is 
formed. 

55.  To  10  cc.  of  the  5  per  cent,  solution  in  a  small  beaker 
add   10  cc.   of  a   saturated   solution   of  ammonium   sulphate.     A 
white  precipitate  of  the  primary  albumoses  is  formed.     Stir  the 
mixture  vigorously  for  a  short  time  with  a  glass  rod  that  has  one 
end  covered  with  a  small  piece  of  rubber  tubing  :  allow  to  stand  for 
a  few  minutes.     The  precipitate  will  usually  gather  together  and 
can  be  almost  completely  collected  as  a  gummy  mass  on  the  end  of 
the  rod.     Transfer  it  to  about  5  cc.  of  hot  water.     The  precipitate 
dissolves.     Cool  the  solution  and  divide  it  into  three  portions. 

(a)  Add  a  drop  of  strong  acetic  acid  and  two  drops  of  potassium 

ferrocyanide.     A  white  precipitate  is  formed,  which  dis- 
appears on  heating  and  reappears  on  cooling. 

(b)  To  another  portion  add  a  few  drops  of  strong  nitric  acid. 

A  white  precipitate  is   formed,   which  disappears   on 
'  heating  and  reappears  on  cooling. 

(c)  To  the  third  portion  add  a  drop  of  copper  sulphate  solution. 

A  white  precipitate  is  formed. 

56.  The  fluid  from  which  the  main  mass  of  primary  albumoses 
has  been  removed  is  filtered  and  treated  in  a  beaker  with  a  single 
drop  of  sulphuric  acid,  and  then  with  ammonium  sulphate  that  has 
been  finely  powdered  in  a  mortar.     The  mixture  is  stirred  vigorously 
till  the  fluid  is  saturated  with  the  salt.     A  flocculent  precipitate  of 
the  secondary  albumoses  (deutero-albumoses)  is  formed.     Collect 
this  ori  the  rod  as  before,  dissolve  in  a  little  water,  divide  the  solution 
into  three  parts,  and  repeat  the  three  tests  already  performed  with 
the  primary  albumoses.    A  precipitate  is  not  formed  by  any  of  the 
reagents. 

57.  The  fluid  from  which  the  secondary  albumoses  have  been 
removed  contains  peptone.     Filter  it,  and  treat  a  portion  of  the 


56  THE    PROTEINS.  [CH.    II. 

filtrate  with  twice  its  volume  of  40  per  cent,  sodium  hydroxide  and  a 
drop  of  i  per  cent,  copper  sulphate.  A  pink  colour  appears,  due  to 
the  presence  of  peptone. 

Important  Note. — This  large  excess  of  strong  NaOH  must  be  added  in 
order  to  decompose  the  (NH4)2SO4  with  which  the  solution  is  saturated.  The 
characteristic  rose  colour  is  only  obtained  when  the  alkalinity  is  due  to  NaOH, 
ammonia  being  quite  inefficient. 

5  cc.  of  saturated  (NH4)2SO4  solution  contains  about  3-75  grms.  of  the  salt. 
This  requires  2-27  grms.  of  NaOH.  10  cc.  of  40  per  cent.  NaOH,  containing 
4  grms.  of  NaOH,  is  thus  sufficient. 

58.  Evaporate  a  small  portion  of  the  original  fluid  to  complete 
dryness,  finishing  the  process  on  a  water  bath  in  order  to  prevent 
charring.     Rub  up  the  residue  with  successive  small  quantities  of 
strong  alcohol  (95  per  cent.).     Add  the  extracts  together,  filter  and 
evaporate  them  to  dryness  on  a  water  bath.     Dissolve  the  residue 
from  this  evaporation  in  a  little  water  and  test  for  proteins  by  the 
various  colour  tests.     Only  insignificant  traces  are  present,  showing 
that  albumoses  and  peptones  are  insoluble  in  strong  alcohol. 

NOTE. — It  is  frequently  desirable  to  remove  all  proteins  from  a  solution 
before  testing  for  certain  substances,  e.g.  sugars,  bile-salts,  urea,  etc.  In  the 
case  of  albumoses  and  peptones  this  can  only  be  effected  by  the  method 
described  above,  advantage  being  taken  of  the  solubility  of  sugars,  etc.,  in 
alcohol,  and  the  insolubility  of  all  proteins  in  the  same.  The  aqueous  solution 
prepared  in  this  way  will  be  spoken  of  as  "  an  alcoholic  extract." 

Peptones.  Use  a  2  per  cent,  solution  of  Savory  and 
Moore's  peptone,  which  is  usually  free  from  albumoses. 

59.  Apply  the  usual  colour  reactions  for  proteins.     They  are 
all  obtained. 

NOTE. — The  glyoxylic  reaction  may  not  be  very  intense,  owing  to  the 
presence  of  chlorides  in  the  preparation.  Pure  peptone,  when  freed  from 
chloride  by  appropriate  means,  gives  a  very  good  glyoxylic  reaction. 

60.  Add  a  drop  or  two  of  strong  acetic  acid  and  a  drop  of 
potassium  ferrocyanide.     No  precipitate  is  produced,  showing  that 
the  primary  albumoses  are  absent. 

61.  Add  a  little  Esbach's  or  Briicke's  solution.     A  very  slight 
or  no  precipitate  is  formed,  if  the  solution  be  free  from  albumoses. 

62.  Saturate  a  portion  with  ammonium  sulphate.     No  precipi- 
tate, or  only  a  slight  one,  is  produced,  showing  that  albumoses  are 
absent. 


CH.    II.]  MUCIN.  57 

63.  Treat  5  cc.  of  the  filtrate  from  Ex.  62  with  two  volumes  of 
40  per  cent.  NaOH  and  a  drop  of  copper  sulphate.     A  pink  colour  is 
formed. 

64.  Add  a  few  drops  of  a  solution  of  tannic  acid.    A  white 
precipitate  is  formed. 

65.  Add  a  few  drops  of  a  solution  of  lead  acetate.     A  white 
precipitate  is  formed. 

J.    The  Gluco-proteins. 

These  bodies  are  conjugated  proteins,  the  protein  being 
united  to  a  carbohydrate  group. 

They  consist  of  the  mucins  and  mucinoids  or  mucoids. 
The  mucins  are  found  in  connective  tissue  and  are  secreted 
by  certain  of  the  salivary  glands  and  various  parts  of  the 
alimentary  canal,  notably  the  large  intestine.  Their 
solutions  are  viscous.  They  are  soluble  in  dilute  alkalies 
and  are  precipitated  from  solution  by  acetic  acid,  the 
precipitate  being  insoluble  in  excess  of  acetic  acid.  They 
are  also  soluble  in  o-i  per  cent,  hydrochloric  acid.  On 
hydrolysis  with  acids  the  sugar  group  is  split  off  and  will 
reduce  Fehling's  solution. 

The  mucoids  are  not  so  viscous  and  not  so  readily 
precipitated  by  acetic  acid,  the  precipitate  being  soluble 
in  excess.  They  are  found  in  ovarian  cysts  and  in  white  of 
egg  [see  Ex.  41  (e)]. 

Preparation  of  Mucin,  Mince  the  submaxillary  gland  of  an  ox,  grind 
with  sand  and  add  o-i  per  cent.  NaOH  (i  litre  to  50  grams  of  the  moist  gland). 
Shake  well  in  a  large  bottle  from  time  to  time  and  leave  for  about  half  an  hour . 
Strain  through  muslin  and  filter  through  coarse  filter-paper.  (This  crude 
solution  should  not  be  prepared  too  long  before  use,  as  mucin  loses  its 
characteristic  properties  if  left  standing  with  alkalies.) 

66.  Add  acetic  acid  drop  by  drop.    A  stringy  precipitate  is 
formed,  insoluble  in  excess  of  the  acid. 

67.  Remove  the  precipitate  on  a  glass  rod,  wash  with  water, 
and  apply  the  usual  colour  reactions  for  proteins,  e.g.  xanthoproteic, 
glyoxylic,  and  Millon's.     They  are  all  given  by  mucin. 


58  THE    PROTEINS.  [cH.    II. 

68.  Treat  some  of  the  precipitate  with  o-i  per  cent.  HC1.    The 
mucin  dissolves. 

69.  Treat  some  of  the  precipitate  with  2  per  cent.  Na2CO3. 
The  mucin  dissolves. 


K.    The  reactions  of  certain  Albuminoids. 

Gelatin  is  found  in  the  body  in  the  form  of  its  anhy- 
dride, collagen.  This  occurs  in  white  fibrous  tissue  and  in 
the  organic  substance  of  bones,  and  can  be  converted  into 
gelatin  by  boiling  with  a  dilute  acid.  Dried  gelatin  swells 
in  cold  water,  but  is  quite  insoluble  in  it.  On  warming,  a 
more  or  less  viscid  solution  is  obtained,  which  solidifies  to  a 
jelly  on  cooling  provided  the  concentration  be  greater  than 
i  per  cent.  This  process  is  reversible  on  warming  and  cool- 
ing. It  is  precipitated  by  half-saturation  with  ammonium 
sulphate,  by  tannic  acid,  phosphotungstic  acid,  Esbach's 
and  Briicke's  reagents,  but  not  by  normal  lead  acetate. 
On  complete  hydrolysis  it  yields  a  high  percentage  of  its 
nitrogen  in  the  form  of  glycine,  but  only  traces  in  the  form 
of  the  aromatic  amino-acids,  tyrosine,  or  trytophane,  and 
none  as  the  sulphur-containing  compound,  cystine.  There- 
fore its  solutions  fail  to  give  the  glyoxylic,  Millon's  and 
sulphur  colour  tests  for  proteins,  and  only  give  a  slight 
xanthoproteic  test,  which  is  due,  either  to  an  impurity  or  to 
a  small  amount  of  phenyl-alanine. 

70.  Break  gelatin  up  into  small  pieces  and  add  a  small  amount 
of  cold  water.  The  gelatin  does  not  dissolve.  Immerse  the  test- 
tube  in  a  beaker  of  boiling  water  and  leave  it  for  a  short  time.  The 
gelatin  dissolves.  Cool  the  tube  under  the  tap  :  the  gelatin  sets  to  a 
jelly.  Perform  the  following  tests  with  an  approximately  I  per 
cent,  solution  of  gelatin : 

(a)  Xanthoproteic  reaction :  slight. 

(b)  Millon's  reaction :  very  slight,  showing  absence  of  tyrosine 

from  gelatin  molecule.     (See  notes  to  Ex.  22.) 

(c)  Glyoxylic    reaction :    not    obtained,    showing    absence    of 

tryptophane.     (Ex.  23.) 


CH.    II.]  GELATIN    AND    KERATIN.  59 

(d)  Biuret  reaction  :  violet  colour. 

(e)  Sulphur  reaction  :  not  obtained,  showing  absence  of  cystine. 

(Ex.  25.) 

(/)  Add  acetic  acid :  no  precipitate. 
(g)  Add  acetic  acid  and  potassium  ferrocyanide :  very  slight  or 

no  precipitate. 

(h)  Add  tannic  acid:  white  precipitate. 

(i)  Add  lead  acetate :  very  slight  or  no  precipitate. 

(j)  Half  saturate  with  ammonium  sulphate.  The  whole  of  the 
gelatin  is  precipitated,  as  shown  by  a  negative  biuret  test 
in  the  nitrate  (distinction  from  peptones). 

(k)  Add  Esbach's  or  Briicke's  solution  :  yellow  or  white  precipi- 
tate (distinction  from  peptones). 

Keratin.  An  insoluble  body  found  in  the  hair,  skin, 
nails,  and  horns.  Remarkable  for  the  high  percentage  of 
cystine  it  yields  on  acid  hydrolysis. 

71.  Perform  the  following  tests  by  using  horn  shavings,  or  hair. 
Note  insolubility  in  hot  or  cold  water,  dilute  acids,  and  dilute 
alkalies. 

(a)  Xanthoproteic  reaction :  well  marked. 

(b)  Millon's  reaction :  well  marked. 

(c)  Glyoxylic  reaction:  well  marked. 

(d)  Biuret  reaction :  not  obtained,  owing  to  insolubility. 

(e)  Sulphur  reaction :  well  marked. 


CHAPTER   III. 

THE  NUCLEOPROTEINS,   NUCLEINS   AND 
NUCLEIC  ACIDS. 

Nucleic  acid  is  a  complicated  organic  acid  containing 
phosphorus,  which  is  found  widely  distributed  in  animal 
and  vegetable  tissues.  It  is  a  special  constituent  of  the 
nuclei  and  is  therefore  most  abundant  in  cellular  organs, 
such  as  the  thymus,  the  pancreas,  the  testis,  and  the 
lymphatic  glands. 

Nucleic  acid  forms  salt-like  combinations,  with  proteins, 
the  amount  and  nature  of  the  protein  combining  with  the 
nucleic  acid  varying  considerably.  Such  combinations  are 
known  as  nucleo-proteins .  They  are  soluble  in  water  and 
dilute  salt  solutions.  They  show  acidic  properties,  being 
soluble  in  alkalies  and  precipitated  by  dilute  acids.  They 
dissolve  to  form  an  opalescent  solution  in  excess  of  strong 
acetic  acid.  (Distinction  from  mucin.) 

A  rather  special  form  of  nucleoprotein  is  nucleohistone , 
in  which  the  nucleic  acid  is  combined  with  the  basic  protein, 
histone.  It  has  similar  physical  properties  to  those  of  the 
other  nucleoproteins,  but  is  precipitated  as  a  calcium  com- 
pound by  0-2  per  cent,  calcium  chloride  solution. 

On  digesting  nucleoprotein  with  pepsin  and  hydro- 
chloric acid,  the  greater  part  of  the  protein  is  removed  as 
peptone,  but  a  certain  amount  is  still  left  combined  with 
the  nucleic  acid.  This  compound  is  known  as  nuclein.  It 
is  insoluble  in  water  and  dilute  salt  solutions,  but  is  soluble 
in  dilute  alkalies. 

By  hydrolysis  of  nuclein  by  pancreatic  juice  or  better  by 
dilute  alkalies,  the  remainder  of  the  protein  is  removed, 
and  there  is  obtained  nucleic  acid. 

Nucleic  acid  is  not  hydrolysed  by  trypsin,  but  it  is 


CH.    III.]  NUCLEIC    ACID.  61 

broken  down  by  a  variety  of  ferments  found  in  the  tissues. 
The  final  products  of  hydrolysis  of  thymus  nucleic  acid  are 

Phosphoric  acid. 

Purine  bases,  adenine,  and  guanine. 

Pyrimidine  bases,  thymine,  and  cytosine. 

An  unknown  hexose  sugar. 

Yeast  nucleic  acid  differs  only  in  yielding  uracil  instead  of 
thymine  and  a  pentose  sugar  (<f-ribose)  instead  of  the  hexose. 
As  to  the  composition  of  the  nucleic  acids,  it  has  been 
established  that  they  consist  of  certain  groups  called 
nucleotides,  which  can  be  liberated  by  the  action  of  enzymes 
found  in  the  tissues,  and  called  nucleotidases.  There  are 
apparently  four  nucleotides  to  the  molecule  of  nucleic  acid. 
The  nucleotides  consist  of  phosphoric  acid-sugar-base,  the 
latter  being  either  a  purine  base  or  a  pyrimidine  base.  By 
the  action  of  an  enzyme,  called  phospho-nuclease,  on  the 
mononucleotides  the  phosphoric  acid  is  split  off,  leaving  the 
carbohydrate  attached  to  the  purine  or  pyrimidine  base. 
These  compounds  are  known  as  purine — or  pyrimidine— 
nucleosides. 

The  nucleotides  can,  however,  be  attacked  by  another 
enzyme,  purine — or  pyrimidine — nuclease,  which  splits  off 
the  base  from  the  phosphoric  acid-sugar  complex. 

The  following  scheme,  suggested  by  Levene  and  Jacobs, 
may  represent  the  structure  of  thymus-nucleic-acid. 

Purine-nucleoside 

Phosphoric  acid— hexose  --guanine 
Phosphoric  acid  — Hexose— thymine 
Phosphoric  acid  —  Hexose — cytosine 

Phosphoric  acid  -  hexose  -  adenine 

Mono-nucleotide. 

For  further  information  on  the  subject  the  student  is 
referred  to  the  valuable  monograph  by  W.  Jones.* 

*  Nucleic  Acids,  by  Walter  Jones.  (Longmans,  Green  &  Co.,  London, 
1914.) 


62 


NUCLEOPROTEINS,  NUCLEINS,  NUCLEIC  ACIDS.   [CH.   III. 


The  purine  bases  are  of  especial  interest  in  connection 
with  the  origin  of  uric  acid. 


(6) 
CH 


(i) 


(7) 
Purine  is  C5H4N4  or  (2)  HC      ($)C  -  NH 


(3)     N 


;CH(8) 


(4)       (9) 


The  figures  in  brackets  are  used  for  indicating  the  position 
of  substitution  groups.  The  chief  purines  of  physiological 
interest  have  either  the  (2),  (6),  or  (8)  H  atoms  replaced,  so 
we  can  write  purine  as 


H...(6)         or  simply 


H 
H 
H 


Adenine  is  6-amino-purine,  PC— — NH2       or 

\H 


N=C.NH2 
HC     C— NH 

I!     II 

N— C— N 


CH 


By  the  action  of  a  deaminising  enzyme,  adenase,  it  is  con 
verted  to  hypoxanthine,  or  6-oxy-purine, 


OH 


Guanine  is  2-amino-6  oxy-purme, 


OH 


CH.    III.]  NUCLEOPROTEIN.  63 

It  is  converted  by  guanase  into  di-oxy-purine,  or  xanthine, 

-OH 


Hypoxanthine,    or    xanthine,    are    oxidised    by    xanthin 
oxydase  into  2,  6,  8  tri-oxy-purine,  or  uric  acid 


< 
OH  (see  page  292). 

OH 

The  pyrimidine  bases  are  less  complicated  than  the 
purine  bases.     The  pyrimidine  ring  is 

(1)  N—  CH     (6) 

(2)  HC     CH      (5) 

II        II 

(3)  N—  CH     (4) 

Uracil  is  2-6-di-oxy-pyrimidine. 

Thymine  is  2-6-di-oxy-  5  -methyl  pyrimidine,  or  5  -methyl 
uracil. 

Cytosine  is  6-amino-2-oxy-pyrimidine. 

Practically  nothing  is  known  as  to  their  behaviour  in 
the  body. 

72.  Preparation  of  nucleoprotein.    Lymphatic  glands  of  the 
ox  or  sheep,  or  the  thymus  of  a  calf  are  freed  from  fat,  finely  minced, 
ground  with  sand  and  extracted  for  twelve  hours  with  ten  times  their 
weight  of  distilled  water  in  a  large  bottle,  a  small  amount  of  toluol  or 
chloroform  being  added  to  prevent  decomposition.    The  bottle 
should  be  shaken  vigorously  at  frequent  intervals  to  break  up  the 
gelatinous  masses  that  sometimes  form.    The  fluid  is  strained  and 
centrifugalised  to  remove  all  debris  (filtration  being  very  slow). 
This  fluid  contains  both  nucleoprotein  and  nucleo-histone. 

73.  To  a  portion  add  dilute  acetic  acid  till  no  more  precipitate 
is  produced,  and  place  on  the  water-bath  at  37°  C.  for  a  few  minutes. 


64  NUCLEOPROTEINS,  NUCLEINS,  NUCLEIC  ACIDS.   [CH.   III. 

A  heavy  precipitate  of  nucleoprotein  and  nucleohistone  is  formed. 
Allow  this  to  settle  in  a  cylinder  :  pour  or  pipette  off  as  much  of  the 
supernatant  fluid  as  possible,  and  filter  the  remainder.  Note  that 
the  precipitate  is  soluble  in  dilute  alkalies  and  is  reprecipitated  by 
acidification ;  that  it  dissolves  to  an  opalescent  solution  in  excess  of 
acetic  acid  (difference  from  mucin) ;  and  that  it  gives  all  the  usual 
colour  reactions  of  proteins. 

74.  To  another  portion  add  one-tenth  of  its  volume  of  2  per 
cent,  calcium  chloride  and  warm  to  37°  C.     A  white  precipitate  of 
nucleohistone  is  formed.     Pour  off  the  supernatant  fluid,  and  to  this 
fluid  add  dilute  acetic  acid  drop  by  drop;  a  white  precipitate  of 
nucleoprotein  is  produced. 

75.  Precipitate  the  nucleoprotein  and  nucleohistone  from  the 
remainder  of  the  fluid  by  means  of  acetic  acid  as  in  Ex.  73.     Collect 
the  precipitate  on  a  filter  paper,  allow  it  to  drain  well,  and  then 
transfer  it  by  means  of  a  spatula  to  a  small  thimble-shaped  porcelain 
capsule.     Heat  carefully,  first  to  drive  off  the  water,  and  then  to 
carbonise  the  residue.     Add  one-third  of  a  crucible  full  of  fusion 
mixture  (K2CO3  two  parts,  KNO3  one  part),  and  heat  as  strongly  as 
possible  till  the  mass  fuses.    Allow  the  melt  to  cool,  and  extract  it 
with  nitric  acid  (diluted  with  an  equal  quantity  of  distilled  water) 
till  the  mixture  no  longer  effervesces.     Filter:  treat  the  filtrate 
with  about  one-tenth  of  its  volume  of  strong  nitric  acid  and  one- 
third  its  volume  of  ammonium  molybdate ;  boil  for  two  minutes. 
A  yellow  precipitate  of  ammonium  phospho-molybdate  separates 
out,  often  on  the  sides  of  the  vessel.     The  phosphorus  of  the  nucleic 
acid  has  been  oxidised  to  phosphoric  acid. 

76.  Preparation  of  thymus  nucleic  acid.     (After  W.  Jones.) 
To  a  boiling  mixture  of  2  litres  of  water,  100  grms.  sodium  acetate  and 
23  grms.  of  caustic  soda,  add  in  small  successive  portions  I  kilo,  of 
trimmed  and  finely  ground  calves  thymus.     Immerse  the  vessel  for 
two  hours  in  boiling  water,  stirring  occasionally.     Dilute  with  one- 
third  volume  of  water  and  make  faintly  but  distinctly  acid  to  litmus 
with  50  per  cent,  acetic  acid.    The  amount  of  acid  required  is 
usually  about  100  cc.,  but  the  final  additions  must  be  made  extremely 
cautiously  until  a  point  is  reached  which  allows  of  good  filtration. 


CH.    III.] 


GUANINE   AND   ADENINE. 


65 


If  a  portion  does  not  filter  well  after  being  boiled  and  dried  on  a  paper 
heated  with  boiling  water,  the  point  must  be  reached  by  the  addition 
of  more  acetic  acid  or  of  caustic  soda.  Now  boil  the  bulk  and  filter, 
using  a  hot  water  funnel.  Concentrate  the  filtrate  on  a  water  bath  to 
about  750  cc.,  and  pour  the  warm  solution  slowly  into  I  litre  of  95 
per  cent,  alcohol  in  a  large  beaker.  Allow  the  mixture  to  stand  over- 
night. The  precipitated  sodium  nucleate  settles  to  a  spongy 
white  mass.  Pour  off  the  .supernatent  fluid  and  squeeze  out  the 
remainder  as  far  as  possible  by  means  of  a  spatula.  Wash  by 
decantation  first  with  80  per  cent.,  and  then  with  95  per  cent. 
alcohol.  Squeeze  out  the  last  wash  fluid  as  much  as  possible  and 
transfer  to  a  flask  with  300  cc.  of  hot  water,  and  heat  on  the  water 
bath  for  30  minutes.  Add  10  cc.  of  20  per  cent,  caustic  soda,  and 
filter  from  insoluble  phosphates,  using  a  hot  water  funnel.  Acidify 
with  acetic  acid  and  pour  into  700  cc.  of  95  per  cent,  alcohol.  Allow 
to  stand  over-night,  wash  by  decantation  with  alcohol  of  increasing 
strength,  and  grind  in  a  mortar  with  absolute  alcohol  until  it  has 
crumbled  into  a  fine  white  powder.  Transfer  to  a  filter  with  absolute 
alcohol  and  dry  in  a  sulphuric  acid  desiccator.  The  product  should 
weigh  over  30  grms.,  and  consists  of  the  soluble  sodium  salt  of 
thymus  nucleic  acid. 

A  4  to  5  per  cent,  solution  in  warm  water  becomes  gelatinous  at 
room  temperature,  the  viscosity  being  decreased  both  by  acetic 
acid  and  sodium  hydroxide. 

77.    Preparation  of  Guanine  and  Adenine  from  Nucleic  Acid. 

Heat  on  a  boiling  water  bath  50  grams,  of  commercial  yeast  nucleic 
acid  for  two  hours  with  200  cc.  of  10  per  cent,  sulphuric  acid  in  a 
flask  fitted  with  a  reflux  condenser.  Treat  the  hot  fluid  with  strong 
ammonia.  Guanine  is  precipitated.  Continue  to  add  the  ammonia 
till  the  neutral  point  is  reached,  and  then  add  an  excess  of  2  per  cent, 
of  the  reagent.  Allow  to  cool  and  filter.  Reserve  the  filtrate  A. 
Wash  the  guanine  with  i  per  cent,  ammonia,  adding  the  washings  to 
A.  Suspend  the  guanine  in  boiling  water  and  dissolve  in  a  minimal 
amount  of  20  per  cent,  sulphuric  acid.  Add  a  small  amount  of 
good  charcoal,  boil,  and  filter.  Add  ammonia  as  before  to  precipi- 
tate the  guanine.  Filter,  dry  at  40°  C.,  and  dissolve  in  20  to  25 
times  its  weight  of  boiling  5  per  cent,  hydrochloric  acid.  Upon 


66  NUCLEOPROTEINS,  NUCLEINS,  NUCLEIC  ACIDS.    [CH.    III. 

cooling  the  solution  deposits  needle  clusters  of  guanine  chloride, 
C5H6N5O.HC1.2H.jO.  The  filtrate  A  is  filtered  again  if  necessary, 
and  made  faintly  acid  with  20  per  cent,  sulphuric  acid.  Boil  and 
add  10  per  cent,  copper  sulphate.  The  adenine  cuprous  compound  is 
precipitated.  Filter  and  wash.  Suspend  in  water  and  decompose 
by  sulphuretted  hydrogen.  Filter  from  copper  sulphide  and 
evaporate  to  dryness  on  the  water  bath.  Dissolve  in  the  smallest 
possible  amount  of  hot  5  per  cent,  sulphuric  acid,  and  allow  to  cool. 
Adenine  sulphate  (C5H5N5)2H2S04.2H2O  is  obtained  in  crystalline 
form. 


CHAPTER   IV. 

THE  PREPARATION  AND  PROPERTIES  OF 
CERTAIN   AMINO-ACIDS. 

Amino-acids  are  compounds  in  which  a  H  atom  of  an 
alkyl  group  of  an  organic  acid  has  been  replaced  by  an 
amino-group. 

CH3  CH2.NH2 

COOH  COOH 

Acetic  acid.          Amino-acetic  acid  (Glycine). 

All  the  physiological  amino-acids  have  the  amino 
group  attached  to  the  same  carbon  atom  as  that  to  which 
the  carboxylic  group  is  attached,  i.e.  they  are  a-amino-acids. 

CH3  CH3  CH2.NH2 

CH2  CH.NH2  CH2 

COOH          COOH  COOH 

Propionic  acid,     a-amino-  fi-amino-propionic  acid, 

propionic  acid 
(alanine). 

The  following  are  the  most  important  amino-acids  that 
have  been  obtained  from  proteins  by  hydrolysis. 

NH2 
GROUP  I.    Neutral  ampholytes      R  -  CH.COOH. 

Glycine  (amino-acetic  acid) 

CH2(NH2).COOH. 


68  PROPERTIES   OF   CERTAIN    AMINO-ACIDS.  [CH.   IV. 

Alanine  (a-amino-propionic  acid) 

CH3.CH(NH2).COOH. 

Valine  (a-amino-iso-valeric  acid) 

•*. 

J>CH.CH(NH2).COOH. 


Leucine  (isobutyl-amino-acetic  acid) 

ru3  ^>CH.CH2.CH(NH2)COOH. 
\^ri3^ 

Phenyl-alanine  C6H5.CH2.CH(NH2)COOH. 

Tyrosine  (/>-oxy-phenyl-alanine) 


(i) 
CH2.CH(NH2)COOH  ......  (4) 

Tryptophane  (^-indol  alanine) 

C8H6N.CH2.CH(NH2)COOH. 

Cystine  (dicysteine  or  di-/3-thio-a-amino  propionic  acid) 

CH2—  S—  S—  CH2 

CH(NH2)  CH(NH2) 

COOH  COOH 

GROUP  II.     Acid  ampholytes.       R  -  COOH. 

CH(NH2).COOH. 

.      Aspartic  acid  CH2.COOH. 

(amino-succinic  acid) 

CH(NH2).COOH. 

Glutaminic  acid  CH2.COOH. 

(amino-glutaric  acid) 

CH2 

CH(NH2).COOH. 


CH.    IV.]  AMINO-ACIDS.  69 

GROUP  III.     Basic  ampholytes. 

Lysine  (a,  e,  di-amino  caproic  acid) 

CH2(NH2).CH2.CH2.CH2.CH(NH2).COOH. 

Arginine  ((S-guanidine-a-amino  valeric  acid) 

MTLT    ^^ 

NH  ^>C-NH-CH2-CH2.CH2.CH(NH2).COOH. 

Histidine  (^-imidazole-alanine) 
CH 
/    \ 
NH     N 

2.CH(NH2).COOH. 


General  reactions  of  the  amino-acids. 

i  .     They  form  two  classes  of  salts  : 

(a)  With  acids,  owing  to  the  presence  of  the 

-  NH2  group  (see  Ex.  80). 

(b)  With  bases,  owing  to  the  presence  of  the 

-  COOH  group  (see  Ex.   79). 

2.  When  dissolved  in  alcohol  and  saturated  with  dry 
hydrochloric  acid  they  form  esters,  which  are  bases  (see 
Ex.  78). 

R.CH.NH2  R.CH.NH2 

+  C2H5.OH  =  +  H20. 

COOH  COOC2H5 

This  reaction  is  of  considerable  importance,  as  Fischer's 
method  of  separation  of  the  amino-acids  is  based  on  the 
fractional  distillation  of  the  esters. 

3.  They  combine  with  aldehydes  to  form  methylene 
compounds,  which  are  acids  (see  Ex.  260). 


70  PROPERTIES   OF   CERTAIN   AMINO-ACIDS.          [CH.   IV. 

R.CH.NH2  R.CH.N  =  CH2 

|  +  H.CHO=  |  +  H20. 

COOH  COOH. 

This  reaction  is  the  basis  of  Sorensen's  method  of  estimating 
the  production  of  amino-acids  during  tryptic  digestion. 

4.  They  form  insoluble  crystalline  compounds  with 
#-naphthalene-sulphochloride. 

SO2C1      H2N.CH.R  qo 

+  =       bU 

COOH 

5.  They  form  moderately  soluble  copper  salts.     With 
the  exception  of  tryptophane  (see  p.  95)  these  can  be  readily 
crystallised  from  boiling  water.    The  tryptophane  copper 
salt  is  characteristically  insoluble,  even  in  boiling  water. 
In  the  presence  of  even  traces  of  other  amino-acids  it  be- 
comes soluble  in  the  solution  of  their  copper  salts. 

6.  They   are   acted   upon   by   nitrous   acid,   yielding 
nitrogen  gas  (see  Ex.  81). 

R.CH.NH2          HNO2  =  R.CH.OH 

|  +  +    N2+H20. 

COOH  COOH 

This  important  reaction  is  the  basis  of  Van  Slyke's  gaso- 
metric  method  for  the  estimation  of  amino-acids  in  blood 
and  tissues. 

7.  They  are  optically  active  with  the  exception  of 
glycine,  which  does  not  contain  an  asymmetric  carbon 
atom  (see  p.  147). 

Methods  of  separation.  The  proteins  can  be  hydro- 
lysed  by  boiling  acids,  boiling  alkalies,  or  by  the  action  of 
certain  enzymes,  e.g.  trypsin.  The  products  are  then 
separated  by  : — 

I.  Fractional  crystallisation  of  the  amino-acids,  or  of 
their  hydrochlorides.  In  this  way  tyrosine, 
leucine,  cystine,  and  glutaminic  acid  hydrochlor- 
ide  are  obtained. 


CH.   IV.] 


AMINO-ACIDS. 


71 


II.  Fractional  precipitation,  i.e.  by  adding  a  reagent 
to  the  mixture  which  forms  an  insoluble  com- 
pound with  only  one  or  a  few  of  the  substances 
present.  In  this  way,  by  the  use  of  mercuric 
sulphate,  tryptophane  was  first  isolated,  and 
cystine  and  tyrosine  can  also  be  obtained  ;  by 
the  use  of  mercuric  chloride  histidine  is  separ- 
ated. 

III.  Fractional  distillation  of  the  esters.  This  method, 
introduced  by  Emil  Fischer,  led  to  the  discovery 
of  several  of  the  amino-acids,  and  serves  for  the 
quantitative  estimation  of  some  of  them. 


Percentage  amounts   of  some  amino-acids  in  certain 
proteins  : 


fc 

g 

* 

!z 

fcS 

J 

ALBUM] 
(Serum 

GLOBUL 

(Serum 

Sf 
3s" 

GLIADI 

(Wheat 

GELATI 

ELASTI] 

KERAFI 
(Horse  ha 

GLOBII 
(Haemoglol 

Giycine 

O 

3'5 

0 

0-4 

16-5 

25-8 

47 



Alanine 

27 

2-2 

0-9 

2-3 

0-8 

6-6 

1-5 

4*2 

Leucine 

20-O 

I87 

10-5 

6-0 

2-1 

21-4 

8-0 

290 

Tyrosine 

2-1 

2>5 

4-5 

1-8 

0 

3'9 

3-2 

1-3 

Tryptophane     .  . 

+ 

+ 

1-5 

i-o 

o 

— 

+ 

+ 

Cystine 

0'3 

07 

o-o 

— 

o 

— 

11-0 

o-3 

Asrartic  acid     .  . 

1-5 

2'5 

1-8 

0-9 

0-6 

+ 

0-3 

4'4 

Glutaminic  acid 

8-0 

8-5 

21-8 

345 

0-9 

0-3 

37 

1-7 

Lysine 

— 

— 

5-8 

o 

3*9 

— 

— 

4*3 

Arginine 

— 

— 

4-8 

3-0 

8-5 

c-3 

— 

5'4 

Histidine 

— 

— 

2-6 

1*2 

0-4 

— 

— 

11-0 

The  figures  in  heavy  type  draw  attention  to  the  reason  why 
certain  proteins  are  used  for  tl  e  preparation  of  particular  amino-acids. 


72  PROPERTIES    OF   CERTAIN    AMINO-ACIDS.  [CH.    IV. 

78.    Glycine. 

A.     The  preparation  of  glycine  ester  hydrochloride  from  gelatin. 

(i.)  To  300  cc.  of  pure  concentrated  hydrochloric  acid  in  a 
round-bottomed  flask  add  100  grams,  of  ordinary  glue. 

(ii.)  Heat  on  a  boiling  water  bath,  with  occasional  shaking,  until 
the  glue  has  dissolved.. 


Fig.  7.     Heating  on  a  sand  bath  under  a  reflux  condenser. 

(iii.)  Boil  the  mixture  on  a  sand  bath  under  a  reflux  condenser 
for  four  hours  (see  fig.  7). 

(iv.)  Transfer  the  dark  product  to  a  litre  distilling  flask,  and 
distil  off  the  acid  as  completely  as  possible  in  vacuo, 
using  the  apparatus  shewn  in  fig.  8.  The  fluid  is  placed 
in  flask  A,  which  is  immersed  in  a  water  bath  maintained 


CH.   IV.] 


GLYCINE. 


73 


at  45°  to  50°  C.  Join  this  flask  to  another  distilling 
flask  B,  by  a  tight-fitting  rubber  stopper.  Connect  the 
side  neck,  D,  of  this  to  an  exhaust  pump,  and  put  the 
pump  in  action.  The  screw  clamp,  C,  is  on  a  piece  of 


Q 


Fig.  8.      Distillation  in  vacua.* 

pressure  tubing  attached  to  a  glass  tube  drawn  to  a  fine 
capillary  that  passes  to  the  bottom  of  the  flask.  The 
screw  must  be  tightened  until  the  bubbles  of  air  pass 
through  the  fluid  at  such  a  rate  that  they  cannot  quite 
be  counted.  The  flask  B  is  cooled  by  means  of  a  stream 
of  cold  water,  which  passes  on  to  it  by  means  of  a  double 
T-piece.  In  the  absence  of  this  it  is  advisable  to  place  a 
piece  of  filter  paper  on  the  flask  to  keep  the  stream  of 
water  well  spread. 

*   For  alternative  method  see  p.  99. 


74 


PROPERTIES   OF   CERTAIN   AMINO-ACIDS.  [CH.    IV. 

(v.)  When  as  much  acid  as  possible  has  been  removed,  slowly 
open  the  screw  C  to  abolish  the  vacuum.  Cautiously  dis- 
connect the  rubber  tubing  at  D  before  turning  off  the 
pump.  With  the  pump  connexions  shewn  in  fig.  9,  the 
abolition  of  the.  partial  vacuum  is  readily  accomplished 
without  danger. 


Fig.  9.      Connexions  of  vacuum  pump  in  author's  laboratory.     With  the  tap 
C  in  the  position  shewn  a  small  amount  of  air  is  admitted  through  the 
•    narrow  tube  A.     When  C  is  turned  through  a  right  angle,  full  suction 
through  E  is  obtained. 

(vi.)  Disconnect  the  apparatus  and  stopper  the  side  neck  of  A 
with  a  cork. 

(vii.)  To  the  thick  viscous  mass  in  the  flask  add  500  cc.  of 
absolute  alcohol  and  heat  on  a  water  bath  under  a  reflux 
condenser  until  it  has  dissolved. 

(viii.)  Allow  the  solution  to  cool  somewhat,  add  3  to  5  grms.  of  a 
good  quality  animal  charcoal,  boil  on  the  water  bath  for 
10  minutes,  and  filter  hot  into  a  flat-bottomed  litre 
flask. 


CH.    IV.] 


GLYCINE. 


75 


(ix.)  Cool  the  filtrate,  first  under  the  tap,  and  then  with  ice,  and 
pass  in  a  stream  of  dry  hydrochloric  acid  gas  (see  fig.  10) 
until  the  fluid  is  saturated. 


Fig.  10.     Apparatus  for  saturating  a  fluid  with  dry 
hydrochloric  acid  gas. 

B  is  concentrated  hydrochloric  acid.  A  is  concentrated  sulphuric^acid, 
which  is  allowed  to  drop  slowly  into  the  flask.  C  contains  concentrated 
sulphuric  acid  to  dry  the  gas.  The  dry  gas  can  be  introduced  into  the 
fluid  by  means  of  a  Folin  absorption  tube,  D,  though  this  is  not  usually 
necessary. 

(x.)  Boil  on  a  water  bath  under  a  reflux  condenser  for  30  minutes, 
cool  thoroughly,  and  allow  it  to  stand  over-night  in  a 
refrigerator.  The  glycine  ester  hydrochloride  generally 
separates  as  a  mass  of  colourless  needles.  Should  this 
not  occur,  it  is  advisable  to  "  sow  "  the  fluid  with  a 
small  quantity  of  the  crystals  obtained  from  another 
preparation,  or  rub  the  sides  of  the  vessel  with  a 
glass  rod.  [A  second  crop  of  crystals  can  often  be  ob- 
tained by  concentrating  in  vacuo  and  repeating  processes 
(ix.)  and  (x.)]. 


76 


PROPERTIES   OF   CERTAIN   AMINO-ACIDS.          [CH.    IV. 


(xi.)  Filter  on  a  small  Buchner  funnel  (fig.  n),  wash  with  a  little 
ice  cold  absolute  alcohol,  and  dry  in  a  desiccator. 

C2H5.OH+HOOC.CH2.NH2.HC1  =  C2H5OOC.CH2.NH2.HC1.+H2O 

Glycine  hydrochloride.          Glycine  ester  hydrochloride. 
Yield:   10  to  15  grams. 

B.     The  conversion  of  glycine  ester  hydrochloride 
into  glycine. 

(i.)  Weigh  the  glycine  ester  hydrochloride, 
place  it  in  a  250  cc.  round-bottomed 
flask  and  add  10  cc.  of  water. 

(ii.)  Add  100  cc.  of  ether  and  cool  in  a 
freezing  mixture. 

(iii.)  Gradually  add  33  per  cent,  caustic 
soda,  shaking  well  during  the  addi- 
tion, until  the  aqueous  layer  is 
neutral  to  litmus.  About  0-8  cc. 
are  required  for  every  gram  of  the 
hydrochloride. 

(iv.)  Add  powdered  potassium   carbonate, 
shaking  vigorously  between  whiles, 
until  the  watery  layer  is  a  paste. 
By  treatment  with  alkali  the  glycine  ester  hydrochloride 
is  converted  into  glycine  ester,  which  is  soluble  in  ether. 

(v.)  Pour  off  the  ether,  filter  it,  and  place  it  in  a  stoppered  flask. 

(vi.)  Add  another  50  cc.  of  ether  to  the  residue,  shake  well, 
remove  and  filter  the  ether,  adding  it  to  that  in  the 
stoppered  flask.  Repeat  this  once  more. 

(vii.)  Dry  the  ether  by  shaking  for  5  to  10  minutes  with  about 
20  grams  of  anhydrous  potassium  carbonate.  Decant 
the  ethereal  solution  and  remove  the  last  traces  of 
water  by  means  of  anhydrous  sodium  sulphate.  This 
is  prepared  by  strongly  heating  25  grams,  of  sodium 
sulphate  in  a  porcelain  dish  and  cooling  the  warm  melt 
in  a  desiccator.  Allow  the  cool,  finely  powdered,  sul- 
phate to  stand  with  the  ether  for  at  least  six  hours. 


Fig.  ii.  Buchner 
funnel  and  filter- 
ing flask. 


CH.    IV.] 


GLYCINE. 


77 


(viii.)  Filter  off  the  ether  and  transfer  it   to  a  distilling  flask. 
Connect  this  to  another  distilling  flask  as  shewn  in  fig.  8. 

(ix.)  Distil  off  the  ether  under  reduced  pressure,  the  receiving 
flask  being  thoroughly  cooled  (best  done  by  packing  with 
ice). 

(x.)  When  all  the  ether  has  been  removed,  change  the  receiving 
flask  and  distil  over  the  glycine  ester,  at  a  temperature  of 
44°  C.  and  a  pressure  of  n  mm.  mercury.  The  receiving 
flask  must  be  well  cooled. 

(xi.)  Transfer  the  distillate  to  a  round-bottomed  flask,  add  10 
times  its  volume  of  water,  and  boil  on  a  sand  bath  under  a 
reflex  condenser  until  the  alkaline  reaction  has  dis- 
appeared. 

(xii.)  Concentrate  the  solution  in  an  evaporating  basin  on  the 
water  bath.  Crystals  of  glycine  are  obtained. 

Properties  of  Glycine.  It  crystallises  from  water  in 
hard,  flattened,  colourless  prisms.  On  heating,  it  becomes 
brown  at  228°,  and  melts  at  2^2°-2^6°.  The  crystals  have 
a  sweet  taste,  from  which  fact  the  original  name  of  glyocoll 
was  derived  (yXvicvs,  sweet  :  xro'XAa,  glue).  It  is  readily 
soluble  in  water  ( i  part  of  glycine  in  4-3  parts  of  cold  water). 
It  is  insoluble  in  absolute  alcohol,  and  in  ether.  When 
boiled  with  concentrated  alkali  ammonia  is  evolved.  On 
treating  the  residue  with  hydrochloric  acid,  hydrocyanic 
acid  is  evolved,  and  oxalic  acid  is  found  to  be  present. 

Aqueous  solutions  give  a  red  colour  with  ferric  chloride, 
similar  to  that  given  by  acetic  acid. 

On  shaking  an  aqueous  solution  with  benzoyl  chloride 
and  sodium  carbonate,  hippuric  acid  is  formed. 

C6H5.COC1  +  H2N.CH2.COOH  =  C6H5.CO.NH.CH2.COOH 

+  HCL 
Benzoyl  chloride.      Glycine.  Hippuric  acid. 

79.  Preparation  of  the  copper  salt  of  glycine.  To  a  solution 
of  about  0-5  gram,  of  glycine  in  about  40  cc.  of  distilled  water, 
add  an  excess  of  freshly  precipitated,  well  washed  cupric  hydroxide. 


78  PROPERTIES    OF   CERTAIN    AMINO-ACIDS.          [CH.    IV. 

Boil  for  5  minutes  and  filter.  Concentrate  the  filtrate  in  an  evaporat- 
ing basin  on  a  boiling  water  bath,  and  set  the  dish  aside.  Fine  blue 
needles  of  the  copper  salt  are  formed,  having  the  composition 

CH2.CH.NH2.COO^ 
CH2.CH.NH2.COO- 

NOTE. — The  copper  hydroxide  is  prepared  by  adding  10  cc.  of  20  per  cent, 
copper  sulphate  to  about  loocc.  of  distilled  water,  and  stirring  in  i6cc.  of 
N.  sodium  hydroxide,  previously  diluted  with  about  300  cc.  of  water.  The 
precipitate  is  filtered  and  very  thoroughly  washed  with  cold  distilled  water 
until  neutral  to  litmus. 

80.    Glutaminic  Acid. 

A.     Preparation  of  Glutaminic  acid  hydrochloride. 

(i.)  To  100  grams,  of  gluten  flour*  in  a  500  cc.  round  bottomed 
flask  add  300  cc.  of  pure  concentrated  hydrochloric  acid, 
and  heat  on  the  water  bath  until  the  gluten  has  dissolved. 

(ii.)  Add  20  grams,  of  good  decolourising  charcoal,  to  remove  the 
dark  "  humin  substance  "  that  is  formed  during  the 
subsequent  hydrolysis. 

(iii.)  Boil  on  a  sand  bath  under  a  reflux  condenser  for  6  hours, 
(iv.)  Dilute  with  an  equal  volume  of  water  and  filter. 

(v.)  Evaporate  the  filtrate  in  vacuo  to  about  150  cc.  (see  pages 
73  and  94). 

(vi.)  Transfer  the  residue  to  a  300  cc.  Erlenmeyer  flask,  cool 
thoroughly,  and  saturate  with  dry  hydrochloric  acid 
gas  (see  fig.  10). 

(vii.)  Allow  the  flask  to  stand  in  the  ice  chest.  After  24  to  48 
hours  a  mass  of  crystals  of  glutammic  acid  hydrochloride 
separates.  Add  an  equal  volume  of  ice  cold  alcohol. 

(viii.)  Filter  on  a  Buchner  funnel  through  a  piece  of  well  washed 
linen  (handkerchief)  cut  to  fit  the  funnel.  Drain  the 
mother  liquor  away  as  completely  as  possible. 

(ix.)  Wash  the  crystals  with  small  amounts  of  ice  cold  concen- 
trated hydrochloric  acid. 

Yield :    about  40  grams. 

*  This  can  be  obtained  from  Messrs.  Bishop  and  Brooke,  21,  Cock  Lane, 
Snow  Hill,  London,  E.G. 


CH.    IV.]  GLUTAMINIC   ACID.  79 

B.  Recrystallisation  of  the  hydrochloride. 

(i.)  Dissolve  the  crystals  in  about  100  cc.  of  water,  boil  with  a 
sufficiency  of  decolourising  charcoal,  and  filter. 

(ii.)  Saturate  the  cooled  filtrate  with  dry  hydrochloric  acid  gas, 
and  allow  to  stand  in  the  ice  chest  over-night. 

(iii.)  Add  an  equal  volume  of  ice  cold  absolute  alcohol  and  filter 
through  linen  on  a  Buchner  funnel. 

(iv.)  Dry  in  a  vacuum  desiccator  over  potash  and  sulphuric  acid, 

C.  Preparation  of  glutaminic  acid  from  the  hydrochloride. 

(i.)  Weigh  the  pure,  dry,  hydrochloride  and  dissolve  it  in  a 
minimal  amount  of  water.  Add  5-44  cc.  of  N.NaOH  for 
every  gram,  this  being  the  amount  required  to  remove 
the  HCl  according  to  the  following  equation. 

HC1 

CH.(NH2).COOH  CH(NH2).COOH 

+  NaOH    =     |  +  NaCl+H2O. 

CH2.CH2.COOH  CH2.CH2COOH 

(ii.)  Evaporate  the  solution  in  vacuo  at  40°  to  50°  to  reduce  the 

volume  to  60  to  100  cc. 

• 
(iii.)  Transfer  the  warm  solution  to  a  beaker,  and  allow  it  to 

stand  over-night  in  the  ice  chest. 

(iv.)  Filter  off  the  crystals  on  a  Buchner,  wash  with  a  little  cold 
water,  and  dry. 

Yield :  18  to  20  grams. 

Properties  of  Glutaminic  Acid.  It  crystallises  from 
water  in  rhombic  tetrahedra,  which  on  rapid  heating 
melt  at  2 1 3°.  It  dissolves  in  about  100  parts  of  cold  water, 
but  is  much  less  soluble  in  alcohol.  Since  it  contains  two 
carboxyl  and  only  one  amino-group,  its  aqueous  solutions 
are  markedly  acid  to  litmus.  The  hydrochloride  forms 


80  PROPERTIES   OF   CERTAIN    AMINO-ACIDS.  [CH.    IV. 

triclinic  tables,  which  melt  at  about  193°.  This  salt  is 
readily  soluble  in  water,  but  only  very  slightly  soluble  in 
concentrated  hydrochloric  acid.  The  calcium  salt  is 
quantitatively  precipitated  by  strong  alcohol,  provided  that 
the  solution  be  sufficiently  concentrated  (25  to  30  per  cent.). 
On  boiling  an  aqueous  solution  of  the  hydrochloride,  it  is 
largely  converted  into  the  internal  anhydride,  pyrollidone 
carboxylic  acid. 

COOH  COOH 


CH.NH2  CH.— NH 

CH2  CH2 

CH9  -  COOH  CH9  -  CO 


H2O 


This  change  does  not  take  place  in  the  presence  of  strong 
hydrochloric  acid,  nor  during  concentration  in  vacua  at 
40°  to  45°.  Glutaminic  acid  is  especially  abundant  in 
vegetable  proteins.  It  is  for  this  reason  that  gluten  flour, 
consisting  of  gliadins  and  glutelins,  is  used  for  its  prepara- 
tion. 

Glutaminic  acid  is  dextrorotatory,  [a]D  in  water  =  +  12°. 
In  10  per  cent,  hydrochloric  acid  [a]D  =  +  31°.' 

81.  Action  of  nitrous  acid.  To  a  solution  of  glutaminic 
acid  in  water  add  a  solution  of  nitrous  acid,  obtained  by  acidifying  a 
strong  solution  of  potassium  nitrite  with  acetic  acid.  An  evolution 
of  nitrogen  gas  occurs. 

COOH  COOH 

CH.NH2  CH.OH 

CH2  +HN02  CH2  +  N2+H20 

CH2.COOH  CH2.COOH 

Oxy-glutaric  Acid. 


CH.    IV.]  ASPARTIC   ACID.  81 

Aspartic  Acid. 

82.    Preparation  from  Asparagine.    Asparagine  is  the  amide  of 
aspartic  acid,  and  it  is  converted  to  the  acid  by  hydrolysis  with  acids. 

HC1 

CH(NH2).COOH.H20  CH(NH2).COOH 

+  2HC1  =       |  +  NH4C1. 

CH2.CONH2  CH2.COOH 

Crystalline  asparagine  Aspartic  acid  hydrochloride 

Mol.Wt.  =  150. 

The  hydrochloride  is  converted  to  aspartic  acid  by  the  addition  of 
the  calculated  amount  of  sodium  hydroxide. 

(i.)  Into  a  500  cc.  round-bottomed  flask  introduce  15  grams,  of 
crystalline  asparagine  (i/io  gm.  mol.)  and  200  cc.  of 
N.HC1  (2/10  gm.  mol.). 

(ii.)  Boil  gently  on  a  sand  bath  under  an  efficient  reflux  con- 
denser for  6  hours. 

(iii.)  Cool  and  add  100  cc.  of  N.NaOH  (i/io  gm.  mol.),  shaking 
during  the  addition.  Set  aside  to  crystallise  in  a  cool 
place.  (This  soda  is  to  convert  aspartic  acid  hydro- 
chloride  into  free  aspartic  acid.) 

(iv.)  Stir  well  and  filter  on  a  Buchner,  and  wash  with  small 
amounts  of  cold  water.  Reserve  the  filtrate  and  wash- 
ings for  obtaining  copper  aspartate,  or  a  further  crop  of 
crystals  by  evaporation. 

(v.)  Recrystallise  by  dissolving  in  the  smallest  possible  amount 
of  50  per  cent  alcohol,  filtering  through  a  hot  water 
funnel,  and  allowing  to  stand  till  quite  cold. 

(vi.)  Filter  on  a  Buchner  and  dry  in  the  air.  Add  the  filtrate  to 
that  obtained  in  (iv.). 

Yield:  about  12  grams,  of  the  recrystallised  product. 


82  PROPERTIES    OF   CERTAIN   AMINO-ACIDS.          [CH.    IV. 

Properties  of  Aspartic  Acid.  It  crystallises  in  small 
rectangular  plates.  It  dissolves  in  about  360  parts  of 
cold  water  and  19  parts  of  boiling  water.  Like  glutami- 
nic  acid,  its  aqueous  solutions  are  markedly  "acid  to  litmus. 
Solutions  in  alkalies  are  laevorotatory,  those  in  hydro- 
chloric acid  are  dextrorotatory. 

The  copper  salt  is  very  characteristic.  It  can  be 
obtained  by  the  method  given  in  Ex.  79,  or  more  readily  by 
boiling  a  solution  with  some  solid  cupric  acetate,  and  filter- 
ing the  hot  liquid.  On  standing,  beautiful  blue  needles 
separate.  This  copper  salt,  which  contains  4!  molecules  of 
water  of  crystallisation,  is  so  sparingly  soluble  that  it 
serves  for  the  estimation  of  aspartic  acid. 

Cystine. 

83.    Preparation  from  hair  (or  wool)  by  Folin's  method. 

(i.)  Heat  500  cc.  of  pure  concentrated  hydrochloric  acid  in  a 
litre  round-bottomed  flask  on  a  water  bath. 

(ii.)  Add  250  grms.  of  human  hair  (the  sweepings  from  a  hair- 
dresser's shop),  or  of  washed  wool  (a  piece  of  a  pure 
woollen  blanket).  If  hair  is  used  it  must  be  added  in 
portions  of  about  50  grams,  at  a  time,  the  hot  mixture 
being  well  agitated  after  each  addition. 

(iii.)  Boil  under  a  reflux  condenser  on  a  sand  bath  for  5  to  6 
hours,  or  until  the  mixture  no  longer  yields  the  biuret 
reaction. 

(iv.)  To  the  hot  mixture  add  solid  sodium  acetate  to  remove  the 
free  hydrochloric  acid.  The  point  is  reached  when  a  drop 
of  the  mixture  added  to  about  2  cc.  of  water  gives  a 
reddish  violet  or  brown,  and  not  a  deep  blue  with  a  few 
drops  of  a  dilute  (0-2  per  cent.)  solution  of  Congo  red. 
Usually  500  to  600  grams,  of  the  solid  are  required.  Allow 
the  mixture  to  stand  over-night. 

(v.)  Filter  on  a  Buchner.  The  precipitate  consists  of  cystine, 
together  with  a  considerable  amount  of  dirt  and  in- 
soluble debris. 


CH.   IV.]  CYSTINE.  83 

(vi.)  Transfer  the  precipitate  to  a  porcelain  beaker  or  dish,  and 
boil  with  150  cc.  of  20  per  cent,  hydrochloric  acid  (by 
volume).  Filter. 

(vii.)  Boil  the  residue  with  another  100  cc.  of  the  hydrochloric 
acid,  filter,  and  mix  the  two  filtrates. 

(viii.)  Boil  these  with  5  grams,  of  good  decolourising  charcoal 
(see  p.  390),  and  filter.  If  the  filtrate  is  not  practically 
colourless,  the  solution  must  be  boiled  with  more  char- 
coal until  a  colourless  or  light  yellow  fluid  is  obtained. 

(ix.)  Add  a  hot,  concentrated,  filtered  solution  of  sodium  acetate 
until  the  free  hydrochloric  acid  is  removed. 

(x.)  Allow  to  stand  till  quite  cold. 

(xi.)  Filter  off  the  cystine,  wash  with  cold  water,  then  with 
alcohol,  and  dry  in  the  air. 

Yield  :  8  to  10  grams. 

Properties  of  Cystine.  It  crystallises  in  characteristic 
hexagonal  plates,  which  are  only  very  slightly  soluble  in 
cold  water  (i  part  in  8840  parts  of  water)  and  in  alcohol. 
It  dissolves  readily  in  mineral  acids,  but  is  insoluble  in 
acetic  acid.  In  normal  acid  urine  it  is  soluble  to  the  extent 
of  i  part  in  2000.  It  is  readily  soluble  in  dilute  alkalies  and 
in  ammonia. 

It  is  precipitated  from  its  solution  in  sulphuric  acid  by 
mercuric  sulphate  (see  p.  90).  On  reduction  it  yields 
cysteine 

CH2.SH. 
CH.NH2 

COOH. 

This  is  readily  oxidised  to  cystine  by  atmospheric 
oxygen  in  ammoniacal  solution. 


84  PROPERTIES   OF   CERTAIN    AMINO-ACIDS.          [CH.    IV. 

84.  Dissolve  a  small  amount  of  cystine  in  one  or  two  cc.  of  5 
per  cent,  sodium  hydroxide.  Add  a  drop  or  two  of  lead  acetate  and 
boil  for  a  minute.  The  solution  is  darkened  owing  to  the  formation 
of  lead  sulphide  (see  Ex.  25). 


Histidine.  HC 


N 


C.  CH2  CH  COOH. 


NH 


CH 


85.    The  preparation  of  histidine  mono-hydrochloride. 

(i.)  Place  i  litre  of  defibrinated  ox  or  sheep  blood  (or  preferably 
I  litre  of  the  centrifuged  corpuscular  mass  from  blood) 
into  a  2-litre,  round-bottomed  flask. 

(ii.)  Add  500  cc.  of  pure  concentrated  hydrochloric  acid,  shaking 
well  during  the  addition. 

(iii.)  Heat  on  a  boiling  water  bath,  shaking  at  intervals,  for  2 
to  3  hours,  until  the  precipitated  blood  proteins  have  re- 
dissolved. 

(iv.)  Boil  on  a  sand  bath  under  a  reflux  condenser  for 
10  hours. 

(v.)  Transfer  to  an  evaporating  basin  and  remove  the  greater 
part  of  the  hydrochloric  acid  by  evaporating  on  a  boiling 
water  bath  in  a  flue  chamber. 

(vi.)  Add  40  per  cent,  caustic  soda  until  the  mixture  is  only 
slightly  acid  to  litmus  paper. 

(vii.)  Allow  to  stand  over-night,  and  filter  on  the  pump.  Wash 
the  precipitate  with  hot  water,  adding  the  washings  to 
the  main  filtrate. 


CH.    IV.]  HISTIDINE.  85 

(viii.)  Place  the  solution  in  an  evaporating  basin,  make  it 
distinctly  alkaline  by  the  addition  of  caustic  soda,  and 
boil  for  30  to  60  minutes  to  remove  ammonia.  This  is 
tested  by  holding  a  moist  litmus  paper  in  the  vapour. 
The  removal  of  ammonia  is  hastened  by  adding  a  small 
volume  of  alcohol. 

(ix.)  Pour  the  solution  into  about  5  litres  of  water  contained  in  a 
large  vessel. 

(x.)  Add  a  hot  saturated  solution  of  mercuric  chloride  in  water 
until  no  further  precipitate  is  obtained,  keeping  the 
solution  sufficiently  alkaline  to  ensure  complete  precipi- 
tation. Usually  about  100  gms.  of  mercuric  chloride 
are  required. 

(xi.)  Allow  the  mercury  compound  of  histidine  to  settle  over- 
night. 

(xii.)  Syphon  off  the  supernatant  fluid. 

(xiii.)  Filter  off  the  precipitate  on  a  large  Buchner  funnel  and 
wash  it  with  cold  water. 

(xiv.)  Transfer  the  precipitate  to  a  porcelain  dish  and  add  hot 
hydrochloric  acid  (25  per  cent,  by  volume)  as  long  as  any 
of  the  precipitate  goes  into  solution,  avoiding  any  large 
excess  of  acid. 

(xv.)  Filter  from  the  insoluble  residue  of  calomel  and  wash  this 
with  cold  water,  adding  the  washings  to  the  bulk  of  the 
fluid. 

(xvi.)  Dilute  the  fluid  to  about  5  litres  with  distilled  water. 

(xvii.)  Dissolve  20  grams,  of  mercuric  chloride  in  water  and  add 
this  to  the  fluid. 

(xviii.)  Make  the  fluid  markedly  alkaline  by  the  addition  of 
caustic  soda. 

(xix.)  Allow  the  voluminous  precipitate  to  settle  over-night. 

(xx.)  Filter  on  the  pump,  drain,  and  wash  thoroughly  by  grind- 
ing with  water  in  a  mortar  and  filter  again. 


86  PROPERTIES   OF   CERTAIN    AMINO-ACIDS.          [CH.    IV. 

(xxi.)  Grind  the  precipitate  with  a  litre  of  water,  transfer  to  a 
flask  and  decompose  by  means  of  sulphuretted  hydrogen 
gas.  As  the  precipitate  is  somewhat  difficult  to  decom- 
pose it  is  necessary  to  pass  the  gas  for  8  to  10  hours.  It 
must  not  be  assumed  the  decomposition  is  complete  as 
soon  as  the  precipitate  has  blackened.  On  no  account 
must  the  solution  be  heated. 

(xxii.)  Filter  off  the  mercuric  sulphide,  wash  it  with  small 
quantities  of  hot  water,  adding  the  washings  to  the  bulk 
of  the  fluid. 

(xxiii.)  Concentrate  the  solution  to  a  syrup  in  an  evaporating 
basin  on  a  boiling  water  bath. 

(xxiv.)  Whilst  still  hot,  add  boiling  97  per  cent,  alcohol,  with 
continuous  stirring,  until  there  is  a  faint  permanent 
turbidity.  Allow  to  stand  over-night. 

(xxv.)  Filter  off  the  crystals  of  histidine  hydrochloride, 
C6H9N302,HC1,H20. 

(xxvi.)  Repeat  (xxiii.)  to  (xxv/)  to  obtain  a  second  crop. 

Recrystallisation. 

(i.)  Dissolve  the  crystals  in  twelve  times  their  weight  of  65  per 
cent,  alcohol,  by  heating  on  a  boiling  water  bath  under  a 
reflux  condenser. 

(ii.)  Add  a  little  charcoal  and  boil  again. 

(iii.)  Filter  through  a  pleated  paper  on  a  hot  water  funnel,  and 
allow  the  filtrate  to  cool.  The  histidine  hydrochloride 
separates  in  the  form  of  beautiful  white  glistening  plates. 

Yield:  6  to  8  grams. 

Properties  o!  histidine.  Histidine  is  readily  soluble  in 
water,  but  very  slightly  soluble  in  alcohol.-  The  aqueous 
solution  has  an  alkaline  reaction.  It  crystallises  from 
alcohol  in  platelets,  which  melt  with  decomposition  at 
about  253°  C. 

The  most  convenient  salt  for  the  isolation  of  histidine 
is  the  monohydrochloride.  It  is  readily  soluble  in  water. 


CH.    IV.]  TRYPTOPHANE.  87 

It  crystallises  from  aqueous  alcohol  in  platelets,  which  melt 
at  251°— 252°  C. 

Even  in  very  dilute  solution  histidine  gives  a  volu- 
minous white  precipitate  with  phosphotungstic  acid.  It 
is  also  precipitated  by  mercuric  chloride  in  alkaline  solution 
and  by  ammoniacal  silver  nitrate  solution. 

86.  Totani's  reaction  for  histidine.    To  a  small  knife  point 
of  the  hydrochloride  in  a  small  beaker,  add  2  cc.  of  10  per  cent, 
sodium  carbonate.     Dissolve  a  rather  larger  quantity  of  diazo- 
benzene-sulphonic  acid  in  4  cc.  of  10  per  cent,  sodium  carbonate  in  a 
test-tube,  and  add  this  to  the  solution  in  the  beaker.     A  dark  red 
colour    is    produced    (Primary    colouration).    Make    the    solution 
distinctly  acid  with  strong  hydrochloric  acid.    The  solution  becomes 
orange  coloured.    Add  zinc  dust  and  allow  the  reduction  to  proceed 
for  about  15  minutes.    Transfer  a  few  cc.  of  the  clear,  colourless, 
supernatent  solution  to  a  test-tube,  and  render  the  solution  alkaline 
by  the  addition  of    25    per    cent,    ammonia.      A    characteristic 
golden  yellow  colouration  is  produced,  which  is  permanent   for 
a  considerable  time  (Secondary  colouration). 

NOTE. — Tyrosine  gives  a  primary  colouration  that  is  identical  with  that 
described  above.  The  secondary  colouration  is,  however,  a  bright  rose  red, 
which  gradually  changes  to  a  reddish  brown.  The  reaction  should  be  tried 
with  the  two  substances  simultaneously. 

Tryptophane. 

87.  Preparation. 

A.     Digestion  of  the  Casein. 

(i.)  Weigh  out  200  grams,  of  commercial  casein.* 

(ii.)  Gradually  stir  this  into  i  litre  of  cold  distilled  water  in  a 
large  beaker,  or  enamelled  vessel,  avoiding  the  forma- 
tion of  lumps  as  far  as  possible. 

(iii.)  Transfer  the  viscous  mass  to  a  Winchester  quart  bottle 
by  means  of  a  large  funnel  with  a  short  wide  neck. 

(iv.)  Wash  out  the  mixing  vessel  with  a  jet  of  hot  water  and 
transfer  this,  to  the  bottle,  washing  the  funnel  down 
with  some  more  hot  water. 

*  "  Laitproto  No.  6  "  (prepared  by  Casein,  Ltd.,  Culvert  Works,  Batter- 
sea,  London,  S.W.)  can  be  recommended  as  a  suitable  preparation. 


88  PROPERTIES   OF   CERTAIN   AMINO-ACIDS.  [CH.    IV. 

(v.)  Add  more  hot  water  to  make  the  volume  up  to  about  2 
litres  and  shake  vigorously. 

(vi.)  Adjust  the  reaction  to  PH  =  about  8-1.  10  cc.  of  the 
mixture  is  taken,  treated  with  about  10  drops  of  cresol 
red  and  titrated  with  0-2  N.NaOH  from  a  microburette 
(or  a  i  cc.  pipette  graduated  in  i/iooth  cc.)  until  a 
reddish  purple  colour  is  obtained.  The  bulk  is  then 
treated  with  the  corresponding  amount  of  N.  soda  (i.e. 
100  times  the  amount  of  0*2  N.  used).  Should  the 
volume  required  exceed  100  cc.,  40  per  cent,  soda  can 
be  used,  this  being  10  N.  The  mixture  is  well  shaken 
at  frequent  intervals  after  the  addition  of  the  soda. 
The  reaction  should  now  be  alkaline  to  cresol  red  and 
acid  to  phenol  phthalein. 

(vii.)  Add  15  cc.  of  toluol  (to  prevent  putrefaction)  and  2  grams, 
sodium  fluoride  dissolved  in  about  10  cc.  of  hot  water 
(to  decrease  the  action  of  oxidases).  Shake  well. 

(viii.)  Add  70  cc.  of  the  pancreatic  extract  described  on  p.  212,  or 
a  corresponding  amount  of  a  commercial  preparation  of 
trypsin  ("  liquor  pancreaticus  ")  and  mix  well. 

(ix.)  Clean  the  inside  of  the  neck  of  the  bottle  with  a  cloth,  insert 
a  cork,  shake  again,  and  stand  the  bottle  in  a  water  bath 
or  air  thermostat  at  37°  to  40°  C.  for  4  days,  shaking  the 
bottle  every  day  without  removing  the  cork. 

(x.)  After  4  days  add  another  50  cc.  of  the  pancreatic  extract, 
and  allow  the  digestion  to  proceed  for  another  4  days, 
making  8  days  in  all. 

(xi.)  Remove  the  bottle  from  the  incubator,  and  allow  it  to 
stand  at  room  temperature  for  24  hours  or  longer. 


B.     Filtration  and  acidification. 

(i.)  Filter  off  the  precipitate,  which  consists  of  tyrosine,  un- 
digested casein,  etc.,  reserving  it  for  the  separation  of 
tyrosine  described  in  Ex.  89. 


CH.    IV.]  TRYPTOPHANE.  89 

(ii.)  Measure  the  filtrate  and  to  every  86  cc.  add  14  cc.  of 
a  50  per  cent,  solution  (by  volume)  of  pure  sulphuric 
acid.  (This  is  prepared  by  gradually  pouring  500  cc.  of 
pure  sulphuric  acid  into  500  cc.  of  distilled  water, 
cooling  thoroughly  under  the  tap  until  the  whole  of  the 
acid  has  been  added.  When  quite  cold  the  volume  is 
made  up  to  1000  cc.  with  distilled  water.)  The  mixture 
now  contains  7  per  cent,  by  volume  of  sulphuric  acid. 

C.     Separation  of  the  mercuric  sulphate  precipitate, 

(i.)  Add  250  cc.  of  a  10  per  cent,  solution  of  mercuric  sulphate 
in  7  per  cent,  sulphuric  acid  (by  volume).  Mix  well,  and 
allow  to  stand  over-night.  (The  mercuric  reagent  is 
prepared  by  grinding  100  grams,  of  mercuric  sulphate 
with  500  cc.  of  distilled  water,  to  which  70  cc.  of  pure 
concentrated  sulphuric  acid  has  been  added,  adding 
distilled  water  to  make  a  volume  of  1000  cc.,  and  filtering 
if  necessary.) 

(ii.)  Filter  off  the  bulky  yellow  precipitate  of  the  mercuric 
sulphate  compound  of  tryptophane  on  a  Buchner  funnel, 
reserving  the  filtrate  for  Ex.  90. 

(iii.)  Wash  the  precipitate  on  the  Buchner  with  cold  5  per  cent, 
sulphuric  acid  (by  volume)  to  remove  the  tyrosine.  It  is 
not  necessary  to  remove  the  tyrosine  completely  at  this 
stage. 

(iv.)  Wash  the  precipitate  with  distilled  water  to  remove  the 
greater  part  of  the  sulphuric  acid. 

D.    Decomposition  of  the  mercuric  sulphate  precipitate. 

(i.)  Transfer  the  precipitate  and  paper  to  a  wide-necked  500  cc. 
flask,  washing  the  remainder  of  the  precipitate  in  the 
Buchner  into  the  flask  by  means  of  about  200  cc.  of 
distilled  water.  Agitate  thoroughly  to  get  a  good  sus- 
pension of  the  precipitate. 

(ii.)  Add  100  cc.  of  boiling  water,  to  which  3  grams,  of  crystalline 
barium  hydroxide  has  been  added.  Shake  well. 


90 


PROPERTIES    OF   CERTAIN    AMINO-ACIDS.  [CH.   IV. 


(iii.)  Test  the  reaction  of  the  solution  by  means  of  litmus  paper. 
If  it  is  still  acid  add  a  hot  solution  of  baryta  until 
alkaline. 

(iv.)  Pass  in  a  stream  of  sulphuretted  hydrogen  gas,  shaking  at 
intervals,  until  the  mixture  is  fully  saturated. 

(v.)  Heat  on  the  water  bath  to  about  50°  C.,  shake  well,  and 
pass  in  more  of  the  SH2  if  the  odour  of  the  gas  is  not 
perceptible. 

(vi.)  Filter  from  the  mixture  of  mercuric  sulphide  and  barium 
sulphate. 

(vii.)  Wash  the  precipitate  with  hot  water,  and  squeeze  the 
paper  in  a  piece  of  muslin.  Filter  these  washings,  etc., 
through  another  small  paper,  and  add  them  to  the  bulk 
of  the  fluid  obtained  in  (vi.). 

viii.)  Add  a  few  drops  of  5  per  cent,  sul- 
phuric acid.  If  a  white  precipitate 
of  barium  sulphate  is  obtained,  it 
indicates  that  an  excess  of  baryta 
had  been  added,  and  that  barium 
sulphide  is  present.  Continue  to 
add  the  dilute  sulphuric  acid  until  no 
further  precipitate  is  obtained.  Filter 
off  the  barium  sulphate.  If  the  addi- 
tion of  the  dilute  sulphuric  acid 
does  not  cause  a  precipitate,  proceed 
directly  to : — 

Remove  the  sulphuretted  hydrogen  by 
a  strong  current  of  air,  using  the 
apparatus  shown  in  fig.  12.  (It  is 
preferable  to  remove  the  SHg  by  dis- 
tillation in  vacuo  at  45°  C.) 


Fig.  12.  Apparatus  for 
removal  of  SH2  by 
means  of  an  air 
current. 


E.    Removal  of  cystine  and  reprecipitation  of  the  tryptophane. 

(i.)  Measure  the  fluid  and  add  14  cc.  of  50  per  cent,  sulphuric 
acid  for  every  86  cc. 


CH.    IV.]  TRYPTOPHANE.  91 

(ii.)  Cautiously  add  the  mercuric  sulphate  reagent  prepared 
as  described  in  C  (iii.)  above.  Add  this  until  a  slight 
definite  precipitate  is  obtained.  Usually  about  15  cc.  are 
required.  Allow  the  mixture  to  stand  for  10  minutes. 
Filter. 

(iii.)  To  the  filtrate  add  80  to  100  cc.  of  the  mercuric  reagent, 
and  allow  the  mixture  to  stand  over-night. 

(iv.)  Filter  on  a  Buchner  funnel,  wash  with  cold  5  per  cent, 
sulphuric  acid,  and  then  thoroughly  with  water. 

(v.)  Suspend  the  precipitate  and  paper  in  50  cc.  of  water. 

(vi.)  Add  2  grms.  of  barium  hydroxide  dissolved  in  70  cc.  of 
boiling  water. 

(vii.)  Decompose  by  SH2  and  proceed  as  in  D  (iv.)  to  D  (ix.), 
taking  care  to  have  only  a  very  slight  amount  of  free 
sulphuric  acid  present. 


F.    Removal  of  sulphuric  acid. 

(i.)  Heat  the  fluid  on  a  boiling  water  bath,  and  add  a  hot  solu- 
tion of  baryta  to  a  point  when  no  further  precipitation 
can  be  seen. 

(ii.)  Filter  a  portion  until  a  clear  filtrate  is  obtained,  and  test 
with  a  drop  or  two  of  the  baryta  solution.  If  no  pre- 
cipitate is  obtained,  test  another  portion  of  the  hot 
nitrate  with  a  drop  of  dilute  sulphuric  acid.  Should  this 
fail  to  give  a  precipitate  also,  the  correct  point  is  reached, 
being  that  at  which  neither  sulphuric  acid  nor  baryta 
gives  a  precipitate.  Baryta  or  sulphuric  acid  must  be 
added  until  this  condition  is  attained,  the  samples  tested 
being  added  to  the  bulk.  The  process  is  much  facilitated 
if  the  relative  concentrations  of  the  alkali  and  aeid  are 
roughly  determined  against  one  another.  Filter  to 
obtain  a  perfectly  clear  nitrate. 

(iii.)  Add  a  single  drop  of  10  per  cent,  ammonia. 


92  PROPERTIES    OF   CERTAIN    AMINO-ACIDS.  [CH.    IV. 

G.    Crystallisation. 

(i.)  Evaporate  in  vacuo  (fig.  8)  at  a  temperature  of  about  45°  C. 
until  the  volume  is  reduced  to  rather  less  than  iocc., 
best  ascertained  by  measuring  10  cc.  into  the  flask  before 
the  evaporation  is  commenced.  (See  page  99.) 

(ii.)  Disconnect  the  apparatus  with  the  usual  precautions. 
(See  page  74.) 

(iii.)  Heat  for  a  short  time  on  a  boiling  water  bath,  shaking  the 
fluid  round  the  flask  to  get  as  much  as  possible  into 
solution. 

(iv.)  Transfer  to  a  small  crystallising  dish  or  beaker  and  add 
an  equal  volume  of  strong  alcohol. 

(v.)  Allow  the  dish  to  stand  in  a  cool  place  over-night, 
(vi.)  Filter  off  the  precipitate,  using  a  suction  pump. 

(vii.)  Wash  out  the  flask  and  dish  with  small  amounts  of 
alcohol  of  increasing  strengths,  65,  75,  85,  and  95  per 
cent.,  using  these  for  washing  the  crystals  on  the  filter. 

(viii.)  Dry  the  crystals  in  the  air. 

H.     Concentration  of  the  mother  liquors. 

(i.)  Evaporate  the  mixed  mother  liquors  and  alcoholic  washings 
in  a  boiling  water  bath,  adding  strong  alcohol  from  time 
to  time,  until  the  crystalline  precipitate  that  forms  at  the 
edge  does  not  redissolve  in  the  body  of  the  fluid  when 
stirred. 

(ii.)  Set  the  dish  aside  for  at  least  an  hour. 

(iii.)  Filter  off  the  crystals  and  wash  with  small   amounts   of 
*  alcohol  of  gradually  increasing  strength. 

(iv.)  Dry  in  the  air. 
Yield:  0-6  to  2  grams. 


CH.    IV.]  TRYPTOPHANE.  93 

I.     Recrystallisation. 

(i.)  Transfer  the  crystals  to  a  small  flask,  fitted  with  a  reflux 
condenser. 

(ii.)  Add  a  small  amount  of  70  per  cent,  alcohol,  and  heat  in  a 
boiling  water  bath.  Very  gradually  add  70  per  cent, 
alcohol  (down  the  stem  of  the  condenser)  until  the 
crystals  have  just  dissolved.  Add  a  large  "knife-point  " 
of  decolourising  charcoal  and  heat  for  five  minutes. 

(iiL)  Disconnect  and  rapidly  filter  through  a  small  hot  funnel 
into  a  small  beaker.  Allow  to  stand  for  at  least  an  hour. 

(iv.)  Filter  on  the  pump,  wash  with  75,  85,  and  95  per  cent, 
alcohol.  Dry  in  a  vacuum  desiccator,  or  in  a  warm  oven 
at  80°  to  90°  C.  There  is  a  considerable  loss  on  recry- 
stallisation. 

Properties  of  Tryptophane.  It  crystallises  from  aqueous 
alcohol  in  white  glistening,  six-sided  plates.  It  is 
moderately  soluble  in  cold  water,  but  freely  soluble  in  hot 
water.  It  is  only  sparingly  soluble  in  absolute  alcohol. 
On  heating  it  changes  colour  at  220°,  browns  at  240°,  and 
melts  at  252°  C.  On  heating  still  further,  there  is  first  an 
evolution  of  carbon  dioxide,  and  then  the  formation  of 
indol  and  skatol. 

Tryptophane  is  optically  active,  being  laevorotatory 
in  aqueous,  but  dextrorotatory  in  acid  or  alkaline  solution. 

It  gives  colour  reactions  with  a  great  variety  of  sub- 
stances. The  most  important  of  these  is  the  reaction  with 
glyoxylic  and  sulphuric  acids  (see  Ex.  23).  The  investiga- 
tion of  the  cause  of  the  similar  colour  reaction  given  by 
proteins  led  to  the  isolation  of  the  amino-acid.  It  also 
gives  colour  reactions  with  most  aldehydes  in  the  presence 
of  strong  hydrochloric  or  sulphuric  acids  containing  a  trace 
of  an  oxidising  substance,  like  ferric  chloride.  All  these 
reactions  are  given  by  tryptophane  when  it  is  combined  in  a 
protein  molecule. 

Free  tryptophane  gives  a  red-rose  colour  when  treated 
with  bromine  water,  the  colouring  matter  being  soluble  in 


94  PROPERTIES   OF  CERTAIN   AMINO-ACIDS.          [CH.   IV. 

amyl  or  butyl  alcohol.      This  reaction  is  not  given  by 
combined  tryptophane. 

Tryptophane  is  somewhat  unstable.  It  is  rapidly 
destroyed  by  boiling  acids,  especially  in  the  presence 
of  carbohydrates,  yielding  a  dark,  so-called  "  humin 
substance,"  or  "  melanin."  It  is  oxidised  by  certain 
metallic  salts,  like  copper  sulphate,  silver  nitrate,  gold 
chloride  and  ferric  chloride,  yielding  indol  aldehyde 


iC.CHO 
HC 

N 

CH     NH 

and  other  substances.  On  heating  on  an  open  water  bath 
in  aqueous  solution  it  suffers  decomposition.  For  this 
reason  it  is  necessary  to  concentrate  in  vacuo,  or  to  add 
considerable  quantities  of  alcohol  during  the  evaporation. 
It  is  much  more  stable  in  alkaline  solutions  and,  in  the 
presence  of  other  amino-acids,  can  be  boiled  with  strong 
baryta  for  days  without  appreciable  loss.  It  must  be 
noted,  however,  that  the  substance  obtained  after  baryta 
hydrolysis  is  racemic  (see  p.  152). 

In  dilute  sulphuric  acid  tryptophane  gives  a  lemon- 
yellow  precipitate  with  mercuric  sulphate,  the  precipitate 
being  a  compound  of  tryptophane  sulphate  with  mercuric 
sulphate.  The  precipitating  effect  of  phosphotungstic  acid 
on  tryptophane  seems  to  vary  with  the  quality  of  the  acid  ; 
some  samples  only  give  a  precipitate  in  concentrated 
solutions,  others  none  at  all,  whilst  some  preparations  pre- 
cipitate it  completely  even  from  relatively  dilute  solutions, 
especially  in  the  presence  of  other  amino-acids.  It  is  not 
precipitated  by  lead  acetate  nor  by  mercuric  chloride  in 
neutral  solution. 

88.  A.  To  a  small  knife  point  of  tryptophane  dissolved  in 
about  3  cc.  of  water,  cautiously  add  bromine  water.  A  rose-red  or 


CH.    IV.l  TYROSINE.  95 

reddish-violet  colour  is  produced,  which  turns  yellow  on  adding  an 
excess  of  bromine.  If  the  addition  of  the  bromine  water  be  stopped 
when  the  red  colour  has  reached  its  maximum  intensity,  it  will  be 
found  that  the  coloured  product  can  be  shaken  out  with  a  few  cc.  of 
amyl  or  butyl  alcohol. 

B.  To  a  small  knife  point  of  tryptophane  add  a  few  cc.  of 
41  reduced  oxalic  acid  "  (see  Ex.  23).    Add  an  equal  volume  of  pure 
sulphuric  acid  and  mix.     A  beautiful  purple  colour  is  produced. 

C.  To  a  small  knife  point  of  tryptophane  add  about  I  cc.  of 
water,  2  drops  of  a  5  per  cent,  solution  of  cane  sugar,  and  5  cc.  of  pure 
hydrochloric  acid.     Boil  for  about  a  minute.     A  deep  purple  solution 
is  obtained. 

D.  Dissolve  about  0-05  gram,  of  tryptophane  in  5  to  10  cc.  of 
water  by  boiling  in  a  test  tube.    Add  as  much  freshly  prepared, 
washed  copper  hydroxide  (see  note  to  Ex.  79)  as  will  go  on  a  large 
spatula  and  boil  for  i  minute.     Filter  hot.     The  nitrate  is  not  blue 
and  gives  no  reactions  for  tryptophane.     The  precipitate  contains 
the  copper  salt  of  tryptophane,  which  is  characteristically  insoluble 
except  in  the  presence  of  even  traces  of  other  amino-acids.     It  is 
then  soluble  in  their  copper  salts. 

Tyrosine. 

Tyrosine  is  the  least  soluble  of  the  amino-acids',  and  for 
that  reason  is  easily  obtained  from  proteins.  When  casein 
is  digested  by  trypsin,  under  the  conditions  described  in 
Ex.  87,  it  generally  happens  that  a  considerable  amount 
separates  as  a  chalky  white  precipitate  in  6  to  10  days. 
The  amount  separating  varies  with  the  activity  of  the 
ferment  preparation,  and  on  the  particular  sample  of  casein 
employed.  If  a  good  yield  of  tyrosine  is  especially  desired 
it  is  advisable  to  concentrate  the  filtrate  obtained  in 
Ex.  87,  B  (i.)  to  about  one-fourth,  allow  to  cool  over-night, 
filter  off  the  precipitate  on  the  pump,  and  purify  by  the 
method  described  below.  The  concentrated  filtrate  can  be 
diluted,  and  used  for  the  isolation  of  tryptophane,  but 
owing  to  its  unstability,  the  yield  of  this  latter  amino- 
acid  is  very  apt  to  be  poor. 


96  PROPERTIES    OF   CERTAIN    AMINO-ACIDS.          [CH.    IV. 

89.  Preparation  of  Tyrosine  from  Casein. 

(i.)  Use  the  mixed  mass  of  calcium  phosphate,  undigested 

casein,  tyrosine,  etc.,  obtained  in  Ex.  87,  B  (i.). 
(ii.)  Boil  the  precipitate  with  about  250  cc.  of  water  to  which 
has  been  added  5  cc.  of  pure  sulphuric  acid.     The  tyro- 
sine dissolves  in  the  acid,  whilst  a  considerable  amount  of 
the  protein  residue  remains  insoluble. 

(iii.)  Filter  through  a  pleated  paper,  passing  the  nitrate  back 
through  the  paper  until  it  is  clear.  Filtration  is  apt  to 
be  rather  slow. 

(iv.)  Heat  the  nitrate  on  a  boiling  water  bath,  and  add  10  cc. 
of  strong  ammonia.  The  reaction  should  now  be  acid  to 
litmus  paper.  Cautiously  neutralise  by  the  addition  of 
ammonia  and  allow  to  cool.  Tyrosine  crystallises  out, 
generally  contaminated  with  calcium  phosphate,  etc. 

(v.)  Filter  off  the  tyrosine  on  the  pump.  Suspend  it  in  about 
300  cc.  of  water  in  a  flask,  boil,  and  add  5  cc.  of  strong 
ammonia  and  boil  for  15  minutes. 

(vi.)  Filter  from  the  calcium  phosphate. 

(vii.)  Neutralise  the  fluid  with  5  per  cent,  sulphuric  acid  and 

allow  to  stand. 

(viii.)  Filter  off  the  tyrosine  on  the  pump,  and  wash  well  with 
.  cold  water.     Wash  with  a  little  alcohol,  and  dry  in  the 
steam  oven  or  in  a  warm  incubator. 

90.  The  separation  of  tyrosine  by  fractional  precipitation. 

(i.)  Treat  the  filtrate  obtained  in  Ex.  87,  C  (ii.)  with  5  volumes 
of  tap  water  in  a  large  vessel,  mix  well,  and  allow  to  stand 
over-night.  Owing  to  the  reduction  in  the  concentration 
of  sulphuric  acid  the  tyrosine  is  precipitated  as  a  com- 
pound with  mercuric  sulphate. 

(ii.)  Syphon  off  the  supernatant  fluid  and  filter  the  precipitate 
on  a  Buchner.  Wash  well  with  water. 

(iii.)  Suspend  the  precipitate  in  about  200  cc.  hot  water  and 
decompose  by  a  stream  of  sulphuretted  hydrogen  gas. 

(iv.)  Boil  and  filter.  Tyrosine  is  left  in  solution  in  dilute 
sulphuric  acid. 


CH.    IV.]  LEUCINE.  97 

(v.)  Neutralise  to  litmus  paper  with  ammonia,  and  allow  to 
stand.  Filter  off  the  crystals  of  tyrosine,  wash  with  cold 
water,  and  dry  in  a  warm  oven. 

Properties  of  tyrosine.  It  crystallises  from  water  in 
white  needles  which  are  characteristically  arranged  in 
sheaves.  It  is  only  very  slightly  soluble  in  cold  water  (i 
part  in  2490  parts  of  water  at  17°  C.),  but  more  soluble  in 
hot  water  (i  part  in  150  parts).  It  is  insoluble  in  ether, 
acetone  and  absolute  alcohol.  It  is  readily  soluble  in 
dilute  alkalies  and  dilute  mineral  acids,  but  it  is  only  very 
slightly  soluble  in  dilute  acetic  acid,  and  practically  insoluble 
in  glacial  acetic  acid.  Its  melting  point  is  rather  indefinite, 
but  on  rapid  heating  is  3  14°  to  3  1  8°.  It  is  laevorotatory  in 
aqueous  acid  and  alkaline  solutions. 

On  being  heated  in  a  tube  it  loses  CO2,  and  is  converted 
to  ^-oxyphenylethylamine.  It  is  not  precipitated  by 
phosphotungstic  acid.  It  is  precipitated  by  mercuric 
sulphate,  the  precipitate  being  soluble  in  dilute  sulphuric 
acid. 

91.  Morner's  reaction.     To  a  few  cc.  of  Morner's  reagent  add 
a  trace  of  tyrosine  and  boil.     A  green  colouration  is  produced. 

NOTE.  —  The  reagent  is  prepared  by  mixing  i  cc.  of  formalin  with  45  cc. 
of  water  and  cautiously  adding  55  cc.  of  strong  sulphuric  acid. 

92.  Millon's  reaction.     To  a  trace  of  tyrosine  add  a  few  cc. 
of  water  and  a  drop  of  dilute  sulphuric  acid  and  boil.     Cool  the 
solution  and  add  a  few  drops  of  Millon's  reagent.     A  precipitate  is 
not  obtained.     Heat.     A  red  colouration  is  obtained. 

NOTE.  —  For  the  preparation  of  Millon's  reagent,  see  Ex.  22.  In  the 
presence  of  5  per  cent,  sulphuric  acid  the  red  colour  is  produced  in  the  cold. 


NH2 


Leucine. 


a-amino-caproic   acid  (a-amino-iso-butyl-acetic   acid). 

Leucine  can  be  obtained  by  fractional  crystallisation  from  a 
protein  digest,  being  separated  by  concentration  of  the 
mother  liquors  left  after  the  isolation  of  tyrosine.  The 


98  PROPERTIES   OF   CERTAIN    AMINO-ACIDS.          [CH.    IV. 

following  exercise  is  suggested  owing  to  the  great  prepon- 
derance of  leucine  over  tyrosine  in  the  proteins  employed. 

93.     Preparation  of  leucine  from  blood. 

(i.)  To  i  litre  of  defibrinated  blood  in  a  2  litre  flask,  gradually 
add  150  cc.  of  pure  sulphuric  acid,  shaking  well  during 
the  addition.  A  semi-solid  mass  of  coagulated  protein  is 
obtained. 

(ii.)  Heat  on  a  boiling  water  bath  for  12  to  16  hours,  shaking 
well  at  intervals. 

(iii.)  Add  some  pieces  of  a  broken  porous  pot,  and  heat  to  boiling 
on  a  large  sand  bath.  It  is  necessary  to  start  with  the 
fluid  hot  from  a  water  bath  and  to  repeatedly  shake  the 
mixture  until  it  boils.  Otherwise  there  is  a  risk  of  the 
flask  breaking.  The  mixture  must  be  boiled  for  10  to 
14  hours. 

(iv.)  To  the  hot  fluid  add  a  hot  solution  of  baryta  until  the 
mixture  is  alkaline  to  litmus.   About  500  grams,  of  baryta 
in  about  i  J  litres  of  boiling  water  are  usually  necessary, 
(v.)  Filter  on  a  Buchner  funnel. 

(vi.)  Make  the  filtrate  acid  to  litmus  with  dilute  sulphuric  acid. 
Concentrate  in  a  porcelain  basin  over  a  free  flame  to 
about  500  cc.  and  filter. 

(vii.)  Render  the  filtrate  faintly  alkaline  to  litmus  by  the 
addition  of  ammonia  and  concentrate  on  a  boiling  water 
bath  until  a  crystalline  crust  has  formed.  Allow  to  cool 
over-night. 

(viii.)  Filter  on  a  small  Buchner  funnel,  pressing  the  mass  of 
crystals  firmly  with  a  pestle  to  remove  as  much  of  the 
mother  liquors  as  possible.  The  filtrate  may  be  further 
concentrated  and  a  second  crop  of  crystals  obtained. 

(ix.)  Recrystallise  from  70  per  cent,  alcohol,  as  described  in 
Ex.  87,  I.  The  product  is  apt  to  be  contaminated  with 
isoleucine 

NH2  NH2 

(  ^  "  >CH.CH.COOH)  and  valine  (™3^  >CH.C1 
\L2.H5-^  /  \L,tl3^ 


CH.   IV.J 


LEUCINE. 


99 


Properties  of  leucine.  Pure  leucine  is  only  very 
slightly  soluble  in  cold  water,  but  when  it  is  contaminated 
with  other  amino-acids  it  is  easily  soluble.  It  is  insoluble 
in  absolute  alcohol,  but  is  soluble  in  hot  dilute  alcohol,  and 
can  be  freed  from  tyrosine  by  crystallising  from  hot  alcohol. 
When  pure  it  crystallises  in  pearly  plates.  When  impure 
it  is  apt  to  crystallise  in  soft  spherical  masses,  which  have  a 
slightly  radiate  structure. 

It  melts  at  297°  with  decomposition.  When  heated 
gently  in  an  open  tube  it  sublimes  at  a  temperature  below 
its  melting  point  and  emits  a  characteristic  smell  of  amyl- 
amine.  It  is  laevorotatory  in  aqueous  solution,  but 
dextrorotatory  in  solution  in  hydrochloric  acid. 


D- 


Topump 


Fig.  13.  Distillation  in  vacuo  by  use  of  a  Claisen  flask  (A).  Alcohol  or  more 
of  the  fluid  can  be  added  through  E  as  the  distillation  proceeds.  D  is  a 
tube  drawn  out  to  a  fine  capillary  through  which  a  small  amount  of  air 
enters  the  boiling  fluid  to  prevent  excessive  bumping. 


CHAPTER  V. 
THE   CARBOHYDRATES. 

There  are  several  groups  of  these  compounds,  only  a 
few  of  which,  however,  are  of  any  physiological  importance. 

A.  The  Monosaccharides,  or  Simple  Sugars. 

B.  The  Compound  Sugars  (Di-  and  Tri-saccharides). 

C.  The   Polysaccharides. 

The  first  two  groups  are  colourless,  crystalline  substances, 
readily  soluble  in  water,  and  usually  of  a  sweet  taste.  The 
polysaccharides  are  mostly  amorphous,  insoluble  in  water, 
and  without  a  sweet  taste. 

A.    The  Monosaccharides. 

A   simple   sugar   is   an   aldehyde,   or   ketone,    linked 
directly  to  at  least  one  alcoholic  group. 

Sugars  containing  the  aldehyde  group  are  known  as 
aldoses,  which  therefore  contain 

H— C— O— H 

H— C  =  O 

as  a  characteristic  group. 

Sugars   containing   the   ketone   group   are   known  as 
ketoses,  of  which  the  group 


H-i 


:— o— H 
=  o 

is  characteristic. 


CH.    V.] 


MONOSACCHARIDES. 


101 


The  simple  sugars  contain  from  two  to  nine  carbon 
atoms,  and  are  called  biases,  trioses,  tetroses,  pentoses, 
hexoses,  etc.,  depending  on  the  number  of  carbon  atoms 
they  contain.* 

The  pentoses,  C5H10O5,  are  widely  distributed  in  nature. 
In  plants  they  are  found  both  in  the  free  state  and  in  the 
form  of  condensation  products,  known  as  pentosans 
(C5H8O4)n.  The  most  important  of  the  pentoses  are  the 
aldoses,  arabinose  and  xylose,  best  obtained  from  the 
corresponding  pentosans  in  gum  arabic  and  beech  sawdust 
respectively. 

Ribose  is  a  constituent  part  of  the  molecule  of  yeast 
nucleic  acid. 

Pentoses  are  occasionally  found  in  human  urine  (see 
P-  312). 

The  hexoses,  C6H12O6,  are  of  great  physiological  import- 
ance. Of  the  many  that  have  been  synthesised  in  the 
laboratory  only  the  following  are  found  in  nature,  and  are 
of  physiological  interest  : — 


CHO 

CHO 

c 

:HO 

CH2OH 

H.C 
H0.( 

:.OH 
:.H 

HO.C 
HO.C 

:.H 
:.H 

H.C.OH 
HO.C.H 

c 

HO.C 

;o 
;.H 

H.C 
H.C 

:.OH 
:.OH 

H.C 
H.C 

:.OH 
:.OH 

HO.C.H 
H.C.OH 

H.C 
H.( 

:.OH 
:.OH 

CH2OH 

CH2OH 

( 

:H2OH 

CH2OH 

d-glucose  : 
(dextrose,  grape- 
sugar) 

d-mannose 

d-galactose 

^-fructose 
(laevulose,    fruit 
sugar) 

*  This  is  not  strictly  true,  for  there  exist  substituted  sugars  in  which  one 
or  more  H  atom  is  replaced  by  a  methyl  group.  A  methyl  pentose  thus  con- 
tains six  carbon  atoms. 


102  THE   CARBOHYDRATES.  [cH.    V. 

It  will  be  noticed  that  the  first  three  are  aldoses, 
whilst  fructose  is  a  ketose. 

The  first  three  are  stereo-isomers,  differing  only  in  the 
arrangement  of  the  H  and  OH  groups  in  space  round  the 
four  central  carbon  atoms,  all  of  which  are  asymmetric. 
(See  p.  151.)  It  therefore  follows  that  these  compounds 
are  optically  active,  that  is,  their  solutions  can  rotate  the 
plane  of  polarised  light.* 

In  the  above  formulae  they  are  represented  as  being 
aldehydes,  but  certain  facts  seem  to  indicate  that  they  can 
exist  in  another  form.  Thus,  if  glucose  be  dissolved  in 
water  it  is  found  that  the  solution  at  first  has  a  much 
higher  rotatory  power  than  when  it  has  been  kept  for 
some  hours  or  has  been  boiled  with  a  trace  of  alkali.  This 
phenomenon  is  known  as  mutarotation.  Also  it  is  very 
much  less  active  chemically  than  the  above  formulas 
warrants. 

These  properties  are  explained  by  assuming  that  when 
first  dissolved  in  water,  glucose  exists  as  a  y-lactone,  having 
the  formulae 

H*C.OH 


H.C 

H.C.OH 
,OH 


CH. 


*  Ordinary  glucose  is  dextro-rotatory.  Fischer  synthesised  its  laevo- 
rotatory  optical  antipode  which  he  called  /-glucose.  All  those  sugars  which 
are  synthetically  related  to  d-glucose  or  d-galactose  he  grouped  into  the  d-  class. 
Thus  ordinary  fructose  he  named  ^-fructose,  though  actually  it  is  strongly 
laevorotatory. 


CH.    V.]  GLUCOSE.  103 

In  this  state  the  *C  atom  is  asymmetric,  so  that  two 
forms  of  glucose  are  possible,  called  a-  and  /3-glucose. 

HO.C.H*  *H.C.OH 


H.C.OH  H.C.OH 


CH2OH  CH2OH 

a-glucose.  ^-glucose. 

Under  certain  conditions  two  forms  of  glucose  can  be 
isolated,  one  with  a  rotatory  power  [a]D  =  +  110°,  the 
other  with  a  rotatory  power  of  [«]D  =  +  19°.  When 
kept  in  solution  there  results  an  equilibrated  mixture  of 
[a]D  =:  +  52'5°-  It  is  possible  that  there  are  three  com- 
pounds now  in  solution,  the  aldehyde  and  the  two 
y-lactones. 

If  the  *H  atom  be  replaced  by  some  other  group 
(generally  aromatic),  the  compound  formed  is  called  an 
a-  or  /5-glucoside,  which  can  be  converted  into  glucose  and 
another  compound  by  hydrolysis  with  acids  or  certain 
ferments. 

The  natural  glucosides  (phloridzin,  salicin,  etc.)  are 
/3-glucosides.  These  glucosides  are  hydro lysed  by  the 
enzyme  emulsin,  which  hydrolyses  all  8-glucosides.  Mal- 
tose (p.  113)  is  glucose-a-glucoside.  It  is  not  hydrolysed 
by  emulsin,  but  is  by  maltase,  which  hydrolyses  all  the 
a-glucosides. 

Physical    and   chemical  properties   of  the   monosaccharides. 

They  are  white  crystalline  solids,  very  soluble  in  water  and 
alcohol.  Insoluble  in  ether,  acetone,  and  most  of  the 
organic  solvents.  Being  adehydes  or  ketones,  they  are 


104  THE    CARBOHYDRATES.  [CH.    V. 

susceptible  of  being  oxidised  to  various  acids,  thus  reducing 
certain  oxidising  reagents.  This  reaction  only  takes  place 
in  hot  alkaline  solutions,  and  is  of  great  value  as  a  test  for 
these  sugars,  and  especially  as  a  basis  of  various  methods 
of  estimation. 

They  react  with  phenyl  hydrazine  in  excess  to  give 
insoluble  crystalline  bodies  called  osazones.  These  are 
of  the  greatest  value  in  determining  the  presence  of  and  in 
characterising  the  monosaccharides,  though  not  in  dis- 
tinguishing them  from  one  another. 

When  heated  with  an  alkali  the  monosaccharides 
become  yellow  and  then  brown,  and  finally  decompose 
into  a  mixture  of  acids  and  resinous  substances. 

They  are  reduced  by  sodium  amalgam  to  hexahydric 
alcohols.  Sorbite  is  formed  from  glucose,  mannite  from 
mannose  and  dulcite  from  galactose.  Fructose  yields  a 
mixture  of  sorbite  and  mannite.  These  alcohols  are  of 
considerable  interest,  as  they  are  used  by  bacteriologists 
for  the  differential  diagnosis  of  certain  pathogenic  organ- 
isms. 

On  oxidation  glucose  gives  rise  to  three  acids — 
CO2H  CHO  CO2H 

I  I  I 

(CHOH)4  (CHOH)4  (CHOH)4 

CH2OH  CO2H  CO2H 

Gluconic  acid.  Glycuronic  acid.    Saccharic  acid. 

Glycuronic  acid  is  interesting  physiologically,  as  it  is 
frequently  found  in  the  urine  in  combination  with  various 
drugs,  such  as  chloral,  camphor,  phenol,  etc.  These  com- 
pounds protect  the  organism  from  the  injurious  effects  of 
the  drugs. 

On  oxidation  galactose  gives  inactive  mucic  acid, 
which  is  isomeric  with  saccharic  acid.  Being  only  slightly 
soluble  its  production  is  used  as  a  test  for  the  presence  of 
lactose  in  urine,  since  lactose  is  hydrolysed  by  acids  into 
galactose  and  glucose. 


CH.    V.]  GLUCOSE.  105 

Glucose  (dextrose  or  grape  sugar)  C6H12O6. 

94.  Preparation  o!  glucose  from  starch. 

To  700  cc.  of  distilled  water  add  40  cc.  of  pure  HC1  and  boil. 
Mix  100  grams,  of  potato  starch  with  200  cc.  of  cold  water,  and 
slowly  stir  this  into  the  boiling  mixture.  Wash  in  the  remainder 
of  the  starch  with  another  100  cc.  of  water.  Boil  under  a  reflux 
condenser  for  3  hours.  To  the  hot  solution  add  solid  lead  carbonate, 
a  little  at  a  time,  till  effervescence  ceases  (about  100  grams,  are 
usually  required).  Cool  and  filter.  Evaporate  the  filtrate  to  a 
thin  syrup.  Add  an  equal  volume  of  hot  95  per  cent,  alcohol. 
Filter.  Evaporate  the  filtrate  to  a  thick  syrup.  Treat  this  with 
twice  its  volume  of  93  per  cent,  alcohol  and  allow  the  solution  to  cool 
slowly.  If  a  syrup  falls  out  of  solution  on  cooling,  the  alcohol  is  too 
strong,  and  a  few  drops  of  water  should  be  added  and  the  solution 
again  heated  to  redissolve  it.  When  the  cooled  solution  no  longer 
deposits  any  syrup  add  a  crystal  of  glucose  and  set  aside  to  crystal- 
lise. 

After  crystallisation  is  complete,  which  may  take  six  or  seven 
days,  drain  the  crystals  and  dry  by  spreading  them  on  a  porous 
earthenware  plate.  To  recrystallise  dissolve  the  dried  crystals  in 
half  their  weight  of  water  and  add  to  the  resulting  syrup  twice  its 
volume  of  boiling  93  per  cent,  alcohol.  Set  the  alcoholic  solution 
aside  to  crystallise,  and  dry  the  resulting  crystals  as  before. 

Unless  directions  to  the  contrary  are  given  use  a  0-2  per  cent,  solution  of 
glucose  for  the  following  exercises. 

95.  Boil  3  cc.  with  I  cc.  of  5  per  cent,  sodium  hydroxide.     The 
solution  turns  yellow. 

NOTE. — The  yellow  colour  is  due  to  the  formation  of  caramel  (a  condensa- 
tion product)  by  the  hot  alkali. 


96.  Treat  two  or  three  cc.  of  5  per  cent,  caustic  soda  with 
four  or  five  drops  of  a  i  per  cent,  solution  of  copper  sulphate.  A 
blue  precipitate  of  cupric  hydroxide,  Cu(OH)2  is  formed.  Add  to 
the  mixture  an  equal  bulk  of  the  sugar  solution.  The  precipitate 
dissolves.  Boil  the  solution  for  a  short  time.  The  blue  colour  dis- 
appears, and  is  replaced  by  a  yellow  or  red  precipitate  of  cuprous 
oxide,  Cu2O  (Trommer's  test). 


106  THE   CARBOHYDRATES.  [CH.   V. 

NOTES.— i.  The  amount  of  copper  necessary  depends  on  the  percentage 
of  sugar  present.  If  only  a  small  amount  of  sugar  be  present  a  mere  disappear- 
ance of  the  blue  colour  is  all  that  may  happen,  or  possibly  the  fluid  may  assume 
a  faint  yellowish-red  tint.  If  excess  of  copper  be  added,  the 'reduction  is 
obscured  by  the  blue  cupric  hydrate  in  solution,  or  the  black  precipitate  of 
cupric  oxide  that  is  formed  on  heating  this  in  the  alkaline  solution.  It  is 
always  best  to  add  the  copper  sulphate  a  few  drops  at  a  time,  boiling  between 
each  addition. 

2.  The  reaction  is  a  type  of  several  that  have  been  introduced  for  the 
detection  of  glucose,  all  of  which  depend  on  the  fact  that  in  alkaline  solution  it 
has  reducing  properties  when  boiled.     For  this  reason,  glucose,  and  all  sugars 
that  have  this  property  are  sometimes  spoken  of  as  "  reducing  sugars." 

3.  The  property  that  glucose  and  other  sugars  have  of  dissolving  cupric 
hydrate  is  common  to  a  large  number  of  organic  compounds,  such  as  glycerol, 
Rochelle  salt  and  sodium  citrate. 

97.  Boil  about  3  cc.  of  Fehling's  solution  (see  Note  i)  in  a 
test-tube.  No  change  occurs.  Add  about  3  cc.  of  the  glucose 
solution  and  boil  again.  A  red  precipitate  of  cuprous  oxide  is 
formed.  (Fehling's  test.) 

NOTES. — i.     Fehling's  fluid  is  prepared  as  follows  : 

(a)  Dissolve  103-92  grams,  of  pure  copper  sulphate  in  warm  distilled 
water  and  dilute  to  one  litre. 

(b)  Dissolve  320  grams,  of  potassium  sodium  tartrate  (Rochelle  salt)  in 
warm  water,  add  a  little  carbolic  acid  to  prevent  the  growth  of  fungi,  dilute  to 
exactly  a  litre  and  filter. 

(c)  Dissolve  150  grams,  of  sodium  hydroxide  in  distilled  water  and  dilute 
to  i  litre. 

For  use  take  exactly  equal  quantities  of  a,  b,  and  c,  and  mix.  Though  the 
individual  constituents  keep  indefinitely,  the  fluid  when  prepared  suffers 
decomposition,  so  that  a  reduction  occurs  on  boiling.  For  this  reason  the  fluid 
should  be  prepared  just  before  use,  and  must  always  be  tested  by  boiling  before 
being  used. 

The  fluid  is  of  such  a  strength  that  the  copper  sulphate  in  10  cc.  is  just 
reduced  by  0-05  grams,  of  dextrose. 

2.  The  addition  of  the  Rochelle  salt  is  for  the  purpose  of  dissolving  the 
cupric  hydroxide  that  would  otherwise  be  precipitated  by  mixing  (a)  and  (c). 

3.  The  test  is  much  more  delicate  and  certain  than  Trommer's  test,  and 
should  always  be  used  in  preference  to  it. 

4.  If  the  fluid  that  is  being  tested  is  acid,  it  should  be  neutralised. 

5.  Ammonium  salts  considerably  interfere  with  Fehling's    test    owing 
to  the  ammonia  liberated  dissolving  the  cuprous  oxide  to  a  colourless   com- 
pound.    If  they  are  present  a  little  extra  alkali  should  be  added,   and   the 
mixture  boiled  for  two  or  three  minutes  to  allow  of  the  evolution   of   the 
ammonia. 

6.  In  testing  for  small  amounts  of  glucose  it  is  advisable  to  avoid  an 
excess  of  Fehling's  solution,  owing  to  the  excess  of  alkali  tending  to  destroy  the 
glucose  before  the  latter  can  exert  its  reducing  reaction  on  the  copper.     The 
neutral  solution  should  be  made  faintly  blue  with  Fehling's  solution,  and  then 
boiled. 


CH.    V.]  GLUCOSE.  107 

98.  To  2  cc.  of  a  i  per  cent,  solution  add  2  cc.  of  40  per  cent, 
sodium  hydroxide.     Heat  to  boiling  and  keep  boiling  for  one  and  a 
half  minutes.     To  the  hot  solution  add  half  its  volume  of  Fehling's 
solution.     No  reduction,  or  only  a  very  slight  one,  is  obtained. 

NOTE. — Glucose  is  completely  destroyed  by  boiling  with  sodium  hydroxide. 

99.  To  3  cc.  of  a  i  per  cent,  solution  add  a  large  "  knife  point  " 
of  anhydrous  sodium  carbonate    Boil  for  I  minute,  cool  under  the 
tap     Add  half  its  volume  of  Fehling's  solution,  and  allow  to  stand 
without  boiling.     The  Fehling's  solution  is  reduced  without  boiling. 

NOTE. — The  experiment  indicates  that  by  the  action  of  alkalies  glucose  is 
converted  to  a  material  that  will  reduce  Fehling's  solution  in  the  cold.  Ex.  98 
indicates  that  this  material  is  destroyed  by  caustic  alkalies.  It  will  be  seen 
later  (Ex.  118)  that  the  disaccharides,  lactose  and  maltose,  differ  from  glucose  in 
that  they  reduce  Fehling's  in  the  cold  after  being  boiled  with  either  sodium 
hydroxide  or  sodium  carbonate. 

100.  To  5  cc.  of  Benedict's  solution  in  a  test-tube,  add  about 
eight  drops  of  the  sugar  solution.     Boil  vigorously  for  one  or  two 
minutes  and  allow  the  tube  to  cool  spontaneously.     The  entire  body 
of  solution  will  be  filled  with  a  precipitate,  red,  yellow,  or  green  in 
colour  depending  on  the  concentration  of  the  sugar.  (Benedict's  test.) 

NOTES. — i.  Preparation  of  Benedict's  solution  for  qualitative  test. 
Dissolve  173  grams,  of  sodium  citrate  and  90  grams,  of  anhydrous  sodium 
carbonate  in  about  600  cc.  of  distilled  water  by  the  aid  of  heat.  Pour  through 
a  folded  filter  and  make  up  to  850  cc.  Dissolve  17-3  grams,  of  crystallised 
copper  sulphate  inioocc.of  water  and  make  up  to  150  cc.  Pour  the  carbonate 
citrate  solution  into  a  large  beaker  and  add  the  copper  solution  slowly,  with 
constant  stirring.  The  mixed  solution  is  ready  for  use  and  does  not  deteriorate 
on  long  standing. 

2.  Benedict's   solution   has    certain   advantages   over   Fehling's.     For 
example,  it  is  not  so  readily  reduced  by  uric  acid  or  urates,  nor  by  creatinine. 
It  is  not  reduced  by  chloroform,  which  is  sometimes  added  to  urine  as  a 
preservative.     It  does  not  destroy  a  small  amount  of  sugar,  as  Fehling's  does 
(see  note  6  to  Ex.  97).     Also  it  can  be  used  for  testing  urines  for  sugar  in 
artificial  light,  since  it  is  the  bulk  and  not  the  colour  of  the  precipitate  that  is  of 
importance. 

3.  Though  Benedict's  test  is  much  better  than  Fehling's  for  the  detection 
of  small  amounts  of  glucose  in  urine,  it  is  not  quite  so  useful  for  other  work. 
The  author  claims  that  his  test  (Ex.  104)  is  the  most  sensitive  for  general  use. 

101.  To  5  cc.  of  the  modified  Barfoed's  reagent  in  a  test-tube 
add  i  cc.  of  the  0-2  per  cent,  solution  of  glucose  and  stand  the  tube 
in  a  beaker  of  boiling  water.     After  three  and  a  half  minutes  remove 
the  tube  and  examine  it  against  a  black  background.     A  definite 
reduction  is  obtained.     Repeat  the  experiment  with  i  cc.  of  the 


108  THE    CARBOHYDRATES.  [CH.   V. 

solution  diluted  I  in  5.  A  reduction  may  or  may  not  be  obtained, 
depending  on  the  sensitiveness  of  the  reagent  (Barfoed's  test, 
Hinkel  and  Sherman's  modification). 

NOTES. — -i.  The  reagent  is  prepared  by  dissolving  45  grams,  of  neutral 
crystallised  cupric  acetate  in  900  cc.  of  distilled  water  and  filtering  if  necessary. 
To  the  filtrate  add  1-2  cc.  of  50  per  cent,  acetic  acid  and  dilute  to  i  litre. 

2.  A  portion  must  show  no  change  when  heated  in  a  boiling  water  bath 
for  10  minutes. 

3.  0-0005  gram,  glucose  generally  gives  the  test,  whereas  0-02  gram,  lactose 
or  maltose,  or  0-03  gram,  sucrose  fail  to  give  the  test. 

102.  Measure  2  cc.  of  a  i  per  cent,  solution  of  glucose  into  a 
test-tube.     Add  3  drops  of  pure  glycerol.     Measure  20  drops  of  a 
20  per  cent,  solution  of  pure  crystalline  copper  sulphate  into  the  tube 
by  means  of  a  dropping  pipette  (see  fig.  5).       Add  2  cc.  of  20  per 
cent,  sodium  hydroxide.     Boil  the  mixture  and  keep  it  boiling  for 
half  a  minute,  shaking  the  tube  during  the  boiling  to  prevent  loss 
by  spurting.     The  addition  of  a  couple  of  glass  beads  helps  smooth 
boiling.      Filter  through  a  small  paper  or  allow  the  tube  to  stand 
in  a  rack  till  the  cuprous  oxide  has  settled.     Repeat  the  experi- 
ment, using  21,  22,  etc.,  drops  of  the  copper  sulphate  if  the  filtrate 
was  yellow ;  19,  18,  etc.,  if  it  was  blue,  until  a  point  is  found  at 
which  an  extra  drop  of  copper  causes  a  change  in  the  nitrate  from 
yellow  to  a  faint  blue. 

NOTE. — The  experiment  gives  one  a  very  rough  method  of  determining 
the  concentration  of  a  solution  of  glucose,  which  can  be  applied  for  finding  the 
approximate  dilution  necessary  when  an  accurate  estimation  has  to  be  made. 
The  reason  for  the  addition  of  glycerol  is  explained  in  Ex.  96,  note  3. 

103.  Measure  2  cc.  of  the  i  per  cent,  solution  into  a  test-tube, 
add  0-5  cc.  of  pure  concentrated  hydrochloric  acid,  and  boil  gently  for 
2  minutes.     Cool  under  the  tap.    Add  the  number  of  drops  of  copper 
sulphate  necessary  to  give  a  faint  blue,  as  found  in  the  preceding 
exercise,  and  three  drops  of  glycerol.     Neutralise  by  the  addition 
of  20  per  cent,  sodium  hydroxide,  the  neutral  point  being  shown  by 
the  appearance  of  a  grey  precipitate.     Now  add  2  cc.  of  20  per  cent, 
sodium  hydroxide,  boil  for  i  minute,  and  allow   to  stand.     An 
increase  in  the  reducing  power  is  not  obtained. 

NOTE. — Compare  the  results  with  those  from  maltose  and  lactose,  Exs. 
120  and  126. 

104.  To  5  cc.  of  the  solution  in  a  test-tube  add  a  large  "knife 
point "  (half  a  gram.)  of  anhydrous  sodium  carbonate.     Shake,  and 


CH.    V.]  GLUCOSE.  109 

heat  to  boiling.  Maintain  active  boiling  for  50  sees.,  shaking  from 
side  to  side  to  prevent  spurting.  Immediately  add  4  drops  of  a 
mixture  of  equal  parts  of  glycerol  and  10  per  cent,  copper  sulphate. 
Shake  for  a  moment  to  mix  and  allow  to  stand  without  further  heat- 
ing for  i  minute.  The  blue  colour  is  discharged,  and  a  yellowish 
precipitate  of  cuprous  hydroxide  forms. 

Repeat  the  experiment,  using  5  cc.  of  the  solution  diluted  I  in 
10  and  i  in  100.  (Cole's  test.) 

NOTE. — The  test  was  elaborated  by  the  author  for  the  detection  of  very 
small  quantities  of  glucose  in  urine  (see  Ex.  381).  It  is  very  sensitive,  and  it  is 
claimed  that  i  part  of  glucose  in  500,000  parts  of  distilled  water  can  be  detected 
by  this  means.  The  instructions  given  are  to  be  strictly  followed.  Many 
samples  of  glycerol  give  a  slight  reduction  when  boiled  with  sodium  carbonate 
and  copper  sulphate,  but  they  do  not  give  a  reduction  when  treated  in  the  way 
described.  The  function  of  the  glycerol  is  to  keep  the  cupric  carbonate  in 
solution. 

105.  To  2  cc.  of  Nylander's  reagent  add  10  cc.  of  the  glucose 
solution,  mix,  and  boil.     Immerse  the  tube  in  a  beaker  of  boiling 
water  for  five  minutes.     A  black  precipitate  of  metallic  bismuth 
separates  out.     (Nylander's  test.) 

NOTES. — i.  Nylander's  reagent  is  prepared  by  dissolving  50  grams,  of 
Rochelle  salt  and  20  grams,  of  bismuth  subnitrate  in  i  litre  of  8  per  cent, 
sodium  hydroxide. 

2.  The  test  is  used  for  detecting  small  amounts  of  glucose  in  urine.  It  is 
superior  for  this  purpose  to  Fehling's  solution  since  it  is  not  readily  reduced  by 
urates,  creatinine,  etc.  The  introduction  of  Benedict's  solution  and  Cole's 
test  have,  however,  led  to  the  disuse  of  Nylander's. 

106.  Treat  2  cc.  of  a  o-i  per  cent,  solution  of  safranine  with 
2  cc.  of  the  glucose  solution  and  2  cc.  of  5  per  cent,  sodium  hydroxide. 
Mix  and  boil,  avoiding  any  shaking.    The  opaque  red  colour  gives 
place  to  a  light  yellow,  owing  to  the  reduction  of  the  safranine  to  a 
"  leuco-base." 

107.  To  3  cc.  of  the  0-2  per  cent,  glucose  add  T  cc.  of  a  solution 
of  sulphindigotate  of  soda  and  a  large  "  knife  point  "  of  anyhdrous 
sodium  carbonate  and  boil.    The  blue  colour  turns  green,  purplish, 
red,  and  finally  yellow.     Shake  with  air  :  the  blue  colour  reappears. 
(Mulder's  test.) 

NOTE. — These  two  experiments  illustrate  the  reducing  properties  of  glu- 
cose in  hot  alkaline  solution.  The  avidity  of  the  reduced  leuco-bases  for 
oxygen  is  shown  by  the  reappearance  of  the  colour  on  cooling  and  shaking 
with  air. 


110  THE   CARBOHYDRATES.  [CH.   V. 

1 08.  To  2  cc.  of  the  solution  add  a  large  "  knife  point  "  of  an- 
hydrous sodium  carbonate  and  a  rather  smaller  amount  of  solid 
picric   acid.     Boil   for  about   a  minute.     A   deep   reddish   brown 
colour  is  produced.     Repeat  the  experiment  with  2  cc.  of  the  solu- 
tion diluted  i  in  10.     A  distinct  colouration  is  produced. 

s*  (N02)3 
NOTE. — Picric  acid.  C6H2^ 

^OH 

/(NO,), 

is  reduced  to  picramic  acid,  C6H2^— -NH2 

\OH. 

by  various  substances  in  alkaline  solution.     The  reaction  serves  as  a  basis 
for  the  colorimetric  method  of  estimation  of  sugar  in  blood  (Ex.  311). 

109.  To  10  cc.  of  the  solution  add  i  cc.  of  strong  acetic  acid. 
Add  as  much  solid  phenyl-hydrazine  hydrochloride  as  will  lie  on  a 
sixpenny  piece,  and  at  least  twice  this  amount  of  solid  sodium 
acetate.     Dissolve  by  warming,  mix  thoroughly,  and  filter  into  a 
clean  tube.     Place  this  in  a  beaker  of  boiling  water  for  30  minutes, 
keeping  the  water  boiling  the  whole  time.     Remove  the  flame  from 
under  the  beaker,  and  allow  the  solution  to  cool  slowly.     A  yellow 
crystalline  precipitate  of  phenyl-glucosazone  appears,  often  before 
the  solution  has  been  heated  for  more  than  20  minutes.     Collect 
some  of  this  by  means  of  a  pipette,  transfer  to  a  slide,  cover  with 
a  slip,  and  examine  under  both  powers  of  the  microscope.     Note 
the  characteristic  arrangement  of  the  fine  yellow  needles  in  fan- 
shaped  aggregates,  sheaves,  or  crosses. 

NOTES. — I.  Glucose  is  an  aldehyde,  and,  like  all  aldehydes  and  ketones, 
forms  a  compound  with  phenyl-hydrazine.  But  this  phenyl-hydrazone  of 
glucose  is  very  soluble,  and  cannot  be  readily  separated.  However,  in  the 
presence  of  an  excess  of  phenyl-hydrazine  at  100°  C.  an  insoluble  osazone  is 
formed. 

CHO  CH :  N.NH.C6H5 

CHOH  C :  N.NH.C6H5 

|  +  3  C6H5.NH.NH2  =  | 

(CHOH)3  (CHOH)3 

CH2OH  CH2OH 

Glucose  Phenyl-osazone  of  glucose 

(phenyl-glucosazone) 
NH3  +  C6H5.NH2 
Aniline. 

2.  Phenyl-hydrazine  is  a  yellow  basic  liquid,  insoluble  in  water,  but 
soluble  in  dilute  acids  to  form  salts.  If  the  base  itself  is  used,  two  or  three 


CH.   V.]  *       FRUCTOSE.  Ill 

drops  should  be  dissolved  in  a  few  cc.  of  strong  acetic  acid,  and  added  to  the 
sugar  solution. 

3.  Phenyl-hydrazine  hydrochloride,   C6H5.NH.NH2.HC1  does   not  give 
an  osazone  when  boiled  with  glucose,  unless  an  excess  of  sodium  acetate  be 
added.     This  acts  on  the  hydrochloride  to  form  phenyl-hydrazine  acetate  and 
sodium  chloride.    In  the  author's  experience  it  is  advisable  to  have  some  free 
acetic  acid  present. 

4.  The  osazone  can  be  recrystallised  as  follows  :  Filter  the  cold  solution 
through  a  small  paper.     Wash  well  with  cold  water.     Boil  a  little  strong  alcohol 
in  a  tube  and  pour  the  hot  solution  on  to  the  paper.     Collect  the  filtrate  in  a 
clean  tube,  boil  it,  and  pass  it  back  through  the  paper.     Repeat  the  process 
until  a  strong  alcoholic  solution  is  obtained.     Heat  it  again,  and  gradually  add 
boiling  water  until  a  faint  turbidity  is  produced.     Heat  again,  add  alcohol 
until  the  solution  is  just  clear  again,  and  then  allow  the  tube  to  cool  slowly. 
Or  the  alcoholic  solution  can  be  concentrated  slightly  on  a  boiling  water  bath. 
The  product  obtained  can  be  filtered,  washed  and  dried  in  a  steam  oven.     The 
melting  point  is  204°  to  205°  C. 

no.  To  about  2  cc.  of  the  solution  add  6  drops  of  a  I  per  cent, 
alcoholic  solution  of  a-naphthol.  To  this  mixture  add  about  2  cc. 
of  strong  sulphuric  acid,  running  it  down  the  side  of  the  tube.  A 
purple  ring  is  formed  at  the  junction  of  the  fluids. 

NOTE. — This  reaction  is  given  by  all  carbohydrates  (see  Ex.  26).  Furfurol 
is  formed  very  readily  from  fructose,  sucrose  and  the  pentoses.  A  modifica- 
tion of  the  test  that  only  succeeds  with  these  sugars  is  given  in  Ex.  114. 

Fructose  (laevulose  or  fruit-sugar)  is  a  keto-hexose. 
It  can  be  prepared  by  the  acid  hydrolysis  of  inulin,  a 
porysaccharide  found  in  the  tuberous  roots  of  the  dahlia, 
dandelion,  Jerusalem  artichoke  and  Inula  Helenium,  from 
which  plant  the  name  inulin  is  derived.  It  can  also  be 
prepared  by  hydrolysing  cane  sugar  with  dilute  acid  and 
separating  the  fructose  from  the  glucose  by  adding  calcium 
hydroxide  to  the  cooled  solution.  Calcium  fructosate 
crystallises  out,  and  can  be  decomposed  by  oxalic  acid. 
It  is  rather  difficult  to  obtain  crystals  of  the  sugar. 

For  the  following  reactions  use  a  dilute  solution  of 
commercial  fructose.  Certain  of  the  reactions  can  be 
demonstrated  by  the  use  of  a  i  per  cent,  solution  of  "invert 
sugar,"  obtained  by  boiling  loocc.  of  i  per  cent,  cane  sugar 
with  i  cc.  of  strong  hydrochloric  acid  for  two  minutes. 

in.     Repeat  Exercises  95  to  97.     They  are  all  obtained. 


112  THE    CARBOHYDRATES.  [cH.    V 

112.  Prepare  the  osazone  as  directed  in  Ex.  109.     It  is  identical 
with  glucosazone. 

NOTE. — The  reaction  between  fructose  and  an  excess  of  phenyl-hydrazine 
is  as  follows  : — 

CH2OH  CH  :  N.NH.C6H5 

c  =  o  c :  N.NH.C.HS 

I  +  3  C6H5.NH.NH2  =  | 

*(CHOH)3  *(CHOH)8 

CH2OH  CH2OH 

+  2  H2O  +  NH3  +  C6H6NH2 

The  configuration  of  the  three  secondary  alcoholic  groups  indicated  by  *  is 
identical  in  glucose  and  fructose  (see  formula  on  page  101).  It  follows  that  the 
osazones  are  identical. 

113.  Repeat  Ex.  no.     It  is  obtained  with  great  brilliance. 

114.  To  about  15  drops  in  a  test-tube  add  6  drops  of  a  i  per 
cent,  alcoholic  solution  of  a-napthol  and  5  cc.  of  concentrated  HC1. 
Boil.     The  solution  becomes  deep  purple  as  soon  as  the  mixture  is 
vigorously  boiling. 

NOTES. — i .  This  modification  of  the  furfurol  test  is  given  only  by  fructose, 
sucrose  and  the  pentoses.  Glucose,  maltose,  lactose,  and  the  common  poly- 
saccharides  only  give  an  intense  colour  after  being  boiled  from  i  to  2  minutes. 

2.  Fructose,  either  in  the  free  state  or  produced  from  sucrose  by  acid 
hydrolysis  and  the  pentoses,  readily  yield  furfurol  (furfuraldehyde)  by  the 
action  of  strong  HC1.     With  H2SO4  furfurol  is  readily  produced  from  all 
carbohydrates  (see  Ex.  no). 

3.  Furfurol  is  HC CH 


HC\         /C.CHO 


O 

It  reacts  with  a-napthol,  thymol,  bile  salts  (see  Ex.  315)  in  the  presence  of 
strong  acids  to  give  coloured  compounds. 

4.  Proteins  that  contain  a  carbohydrate  group  also  give  a  reaction 
(see  Ex.  26). 

115.  Seliwanoff's  test  for  fructose.  To  5  cc.  of  Seliwanoff's 
reagent  add  a  few  drops  of  the  sugar  and  heat  the  solution  to  boiling. 
A  red  colouration  and  a  red  precipitate  are  formed.  The  precipitate 
dissolves  in  alcohol,  to  which  it  imparts  a  striking  red  colour. 

NOTES. — The  reagent  is  prepared  by  dissolving  0-05  gram,  of  resorcin  in 
100  cc.  of  hydrochloric  acid,  diluted  with  its  own  volume  of  water. 

The  test  is  also  given  by  glucose  after  long  boiling,  but  a  precipitate  is  not 
usually  formed. 


CH.   V.]  MALTOSE.  113 

B.    The  Disaccharides. 

Maltose  is  the  disaccharide  formed  as  the  final  product 
of  the  hydrolysis  of  starch  by  enzymes,  such  as  ptyalin, 
diastase,  etc.  It  is  hydrolysed  by  boiling  acids,  and  by 
the  enzyme  maltase  of  the  small  intestine,  to  two  molecules 
of  glucose.  It  exhibits  well-marked  reducing  properties 
towards  Fehling's  solution,  but  not  towards  Barfoed's. 
It  forms  an  osazone  with  phenyl-hydrazine  acetate,  which 
is  more  soluble  than  glucosazone  and  which  melts  at 
2o6°C.  Constitutionally  it  is  glucose-a-glucoside. 

116.    Preparation  of  Maltose. 

Weigh  out  200  grams,  of  fine  potato  starch  and  divide  it  into 
three  approximately  equal  portions.  Add  50  cc.  of  cold  water  to  one 
portion  and  stir  until  a  uniform  cream  is  obtained.  Pour  this 
slowly  into  1200  cc.  of  boiling  water  contained  in  an  enamelled  iron 
vessel,  stirring  well  during  addition.  Boil  for  a  minute,  stirring 
all  the  time.  Cool  to  55  C.  and  add  2  cc.  of  a  fresh  malt  extract 
(see  note  i). 

The  starch  paste  becomes  liquified  in  a  few  minutes.  Boil  the 
liquid  again,  and  to  it  add  the  second  portion  of  starch,  which  has 
been  stirred  up  with  another  50  cc.  of  cold  water.  Cool  to  55  C., 
add  2  cc.  of  malt  extract,  and  liquify  as  before.  Boil  again,  add  the 
third  portion  of  starch,  and  cool  to  55  C.  Add  40  cc.  of  malt  extract, 
and  digest  for  24  hours.  Boil,  filter,  and  evaporate  in  a  porcelain 
dish  on  a  water  bath  until  a  fairly  thick  skim  forms  on  the  surface. 
On  another  water  bath  heat  500  cc.  of  95  per  cent,  alcohol  in  a  flask 
and  pour  it  on  to  the  hot  syrupy  solution,  stirring  well.  The 
maltose  dissolves  and  the  dextrin  is  precipitated,  carrying  down 
with  it  a  considerable  percentage  of  the  maltose.  Connect  the 
flask  to  a  reflux  condenser  (fig.  7)  and  heat  on  a  boiling  water 
bath  for  5  hours,  repeatedly  agitating  the  mixture.  Allow  the 
mixture  to  cool  thoroughly,  and  pour  off  the  alcoholic  solution 
of  maltose  from  the  gummy  residue  of  dextrine.  Transfer 
the  alcoholic  solution  to  a  distilling  flask  connected  with  a 
condenser  and  distil  off  the  alcohol  as  completely  as  possible  by 
heating  the  flask  on  a  water  bath.  Pour  the  thin  syrupy  residue 


114  THE   CARBOHYDRATES.  [CH.   V. 

into  a  beaker  and  allow  to  cool.  Add  a  little  crystalline  maltose 
and  allow  to  stand  for  24  hours  in  a  cool  place.  The  syrup  should 
set  to  a  semi-solid  crystalline  mass.  Spread  this  on  a  porous  earthen- 
ware plate  to  dry. 

Recrystallisation. 

Weigh  the  solid,  add  one-fourth  of  its  volume  of  water,  and 
heat  on  the  water  bath  until  a  syrup  is  formed.  For  every  cc. 
of  water  taken  add  10  cc.  of  hot  88  per  cent,  alcohol.  Filter.  Cool, 
add  a  little  crystalline  maltose,  and  allow  to  stand  for  about  2  days. 
Filter  on  the  pump,  wash  with  a  little  95  per  cent,  alcohol,  and  dry 
on  a  porous  plate. 

NOTES.— i.  Preparation  of  malt  extract.  Mix  40  grams,  of  finely  ground 
pale  dried  malt  with  100  cc.  of  cold  water.  Shake  well,  and  allow  to  stand  for 
lour  hours.  Filter. 

2.  It  is  easier  to  prepare  a  strong  solution  of  starch  by  using  soluble 
starch  (see  p.  391).  In  this  case  boil  1200  cc.  of  water,  stir  200  grams,  of  the 
soluble  starch  with  200  cc.  of  cold  water,  and  pour  this  slowly  into  the  water, 
kept  hot  on  a  boiling  water  bath.  Cool  to  55°  C.  and  add  40  cc.  of  the  malt 
extract. 

Use  a  i  per  cent,  solution  for  the  following  exercises : — 

117.  Repeat  Exs.  95,  99  and  100.     The  reactions  are  indis- 
tinguishable from  those  of  glucose. 

118.  Repeat  Exercise  98.     A  reduction  is  generally  obtained. 
(Distinction  from  glucose.) 

119.  Repeat  Exercise  102,  using  12  drops  of  the  20  per  cent, 
copper  sulphate  for  the  first  trial.     It  will  be  seen  that  the  maltose 
has  a  reducing  power  of  about  60  per  cent,  that  of  a  glucose  solution 
of  the  same  strength. 

120.  Repeat  Exercise  103,  using  20  drops  of  the  copper  sul- 
phate for  the  first  trial.     The  reducing  power  of  the  solution  has  been 
markedly  increased,   owing  to  the  hydrolysis  of  the  maltose  to 
glucose.     (Distinction  from  glucose.) 

121.  Repeat  Exercise  101.     A  reduction  is  not  usually  ob- 
tained.    (Distinction  from  glucose.) 

NOTE. — It  must  be  remembered  that  the  glucose  solution  employed  was 
0-2  per  cent.  Two  cc.  of  this  can  only  reduce  4  to  5  drops  of  20  per  cent, 
copper  sulphate  (see  Ex.  102).  So  that,  though  the  i  per  cent,  maltose  solu- 
tion employed  exhibits  strong  reducing  powers  towards  alkaline  copper 


CH.    V.]  LACTOSE.  115 

solutions,  it  has,  relatively,  a  very  feeble  reducing  power  towards  the  acid 
Barfoed's  reagent.  It  must  be  emphasised  that  useful  information  can  only 
be  derived  from  Barfoed's  test  if  the  reducing  power  of  the  solution  towards 
alkaline  reagents  is  known. 

122.  Prepare  the  osazone  as  directed  in  Exercise  109.     Malt- 
osazone  is  much  more  soluble  than  glucosazone,  and  only  separates 
on  cooling.     It  is  important  to  allow  the  solution  to  cool  slowly  as 
directed.     It  generally  crystallises  in  clusters  of  broad  plates,  not  in 
needles.     It  can  be  recrystallised  by  dissolving  the  precipitate  in 
boiling  water,  filtering,  and  allowing  the  hot  solution  to  cool  slowly. 
It  melts  at  206°  C. 

Lactose  is  the  sugar  found  in  milk,  and  often  in  the 
urine  of  women  during  lactation.  It  has  reactions  very 
similar  to  those  of  maltose.  It  is  hydrolysed  by  boiling 
acids,  and  by  the  ferment  lactase  into  equal  parts  of  glucose 
and  galactose. 

Constitutionally   it   is   glucose-/3-galactoside. 
It  is  not  fermented  by  ordinary  yeast. 

Use  a  i  per  cent,  solution  for  the  following  exercises : — 

123.  Repeat  Exs.   95,   97,   99  and  100.     The  reactions  are 
indistinguishable  from  those  of  glucose. 

124.  Repeat   Ex.    98.     A    reduction   is   generally   obtained. 
(Distinction  from  glucose.) 

125.  Repeat  Ex.  102,  using  14  drops  of  the  20  per  cent,  copper 
sulphate  for  the  first  trial.     Lactose  has  a  reducing  power  about 
70  per  cent,  of  that  of  glucose. 

126.  Repeat  Ex.  103,  using  18  drops  of  the  copper  sulphate 
for  the  first  tube.     The  reducing  power  of  the  solution  has  been 
markedly  increased,  owing  to  the  hydrolysis  of  the  lactose  to  glucose 
and  galactose.     (Distinction  from  glucose.) 

127.  Repeat  Ex.  101.     A  reduction  is  not  usually  obtained. 
(Distinction  from  glucose.) 

128.  Prepare  the  osazone  (see  Ex.  108).     Lactosazone  is  much 
more  soluble  in  hot  water  than  glucosazone.     It  crystallises  in 
irregular  clusters  of  fine  needles  ("  Hedge-hog  crystals  ").     It  can 
be  recrystallised  from  hot  water  (see  Ex.  122).     It  melts  at  200°  C. 


116 


THE    CARBOHYDRATES. 


[CH.    V. 


Sucrose  (cane  sugar)  is  widely  distributed  in  the 
vegetable  kingdom,  where  it  functions  as  a  reserve  material. 
It  crystallises  well,  is  very  soluble  in  water,  and  has  a  much 
sweeter  taste  than  glucose. 

It  does  not  reduce  Fehling's  solution,  does  not  form  an 
osazone,  and  does  not  behave  as  an  aldehyde  or  ketone. 
It  is  hydrolysed  very  readily  by  boiling  acids  to  a  mixture 
of  glucose  and  fructose.  Cane  sugar  is  dextrorotatory, 
but  since  fructose  is  more  laevorotatory  than  glucose  is 
dextrorotatory,  a  mixture  of  the  two  in  equal  parts  is 
laevorotatory.  So  the  sign  of  rotation  being  inverted  by 
hydrolysis,  the  process  is  known  as  inversion,  and  the 
product  as  "  invert  sugar."  This  hydrolysis  is  also  effected 
by  the  enzyme  invertase  (sucrase),  which  is  found  in  the 
small  intestine  and  in  certain  yeasts. 

The  constitution  of  cane-sugar  is  not  yet  definitely 
established,  but  in  all  probability  it  is  formed  by  the 
condensation  of  glucose  and  fructose  in  such  a  way  as  to 
destroy  both  the  aldehyde  and  the  ketone  groups. 


H.C 


CH2OH 


CH2OH 

Glucose  portion. 


CH8OH 

Fructose  portion. 


Use  a  freshly  prepared  i  per  cent,  solution  of  pure  white  crystalline  cane 
sugar  ("  coffee  sugar  ")  for  the  following  reactions. 

129.     Repeat  Exs.  95  and  97.     Sucrose  is  not  affected  by  alkali, 
and  does  not  reduce  alkaline  copper  solutions. 


CH.    V.]  STARCH.  117 

130.  To  3  cc.  of  the  solution  add  i  drop  of  concentrated  HC1- 
Boil  for  a  few  seconds.     Cool  under  the  tap.     Add  about  10  drops  of 
20  per  cent,  copper  sulphate,  3  drops  of  glycerol,  and  about-  3  cc.  of 
20  per  cent,   sodium  hydroxide.     Boil.     A   marked  reduction  is 
obtained. 

NOTE. — Sucrose  is  hydrolysed  extremely  rapidly  by  acids  into  glucose  and 
fructose.  Though  the  polysaccharides  yield  reducing  sugars  by  acid  hydrolysis 
the  above  procedure  would  have  very  little  effect  on  them. 

131.  Repeat  Exs.  114  and  115.     Sucrose  behaves  like  fructose. 


C.    Polysaccharides. 

These  compounds  are  formed  by  the  condensation  of  an 
indefinite  number  of  molecules  of  monosaccharides. 

The  pentosans  (C5H8O4)n  yield  pentoses  on  hydrolysis. 

The  hexosans  (C6H10O5)n  yield  hexoses,  generally  glu- 
cose, on  acid  hydrolysis.  The  polysaccharides  described 
below  are  hexosans. 

Starch  is  widely  distributed  in  the  vegetable  kingdom 
as  a  reserve  carbohydrate.  It  occurs  in  the  form  of  grains 
in  many  roots,  tubers,  seeds,  and  leaves.  The  size  and 
shape  of  the  grains  are  peculiar  to  each  botanical  species. 

These  grains  probably  consist  of  at  least  two  substances. 

A.  "  Amylopectin,"    or    "  starch    cellulose." 

B.  "  Amylose,"    or    "  granulose." 

Amylopectin  forms  about  60  per  cent,  of  the  grain.  It 
is  a  mucilaginous  substance,  which  swells  up  without  dis- 
solving in  boiling  water  or  in  cold  sodium  hydroxide.  It  is 
hydrolysed  by  acids  into  glucose.  By  certain  enzymes, 
called  amylases,  found  in  malt,  saliva,  and  the  pancreas,  it  is 
converted  into  a  mixture  of  maltose  and  "  stable  dextrin," 
that  is  only  very  slowly  hydrolysed  to  maltose  and  glucose. 
It  does  not  seem  to  give  a  blue  colour  with  iodine. 

Amylose  is  soluble  in  cold  water.  It  is  rapidly  and 
completely  converted  to  maltose  by  the  amylases  without 
leaving  a  residuum  of  dextrine.  It  gives  a  blue  colour  with 


118  THE   CARBOHYDRATES.  [cH.    V. 

iodine.  The  grains  are  covered  with  a  film  of  amylopectin, 
which  prevents  the  amylose  from  dissolving  out  in  cold 
water. 

"  Starch  paste  "  is  obtained  by  pouring  a  suspension  of 
the  grains  in  cold  water  into  boiling  water.  It  is  to  be 
considered  as  a  mixture  of  amylopectin  and  amylose,  both 
of  the  substances  being  colloids.  It  is  opalescent,  due  to 
the  amylopectin.  It  is  completely  precipitated  by  half 
saturation  with  ammonium  sulphate,  or  by  the  addition 
of  an  equal  volume  of  strong  alcohol.  It  has  no  reducing 
properties. 

"Soluble  starch  "  differs  from  starch  paste  in  being  clear 
and  limpid.  It  is  produced  by  the  action  of  amylases  or 
acids  on  starch  paste.  It  is  only  slowly  precipitated  by 
half  saturation  with  ammonium  sulphate,  but  is  precipi- 
tated immediately  by  full  saturation.  It  has  no  reducing 
properties. 

Dextrins  are  formed  by  the  partial  hydrolysis  of  starch 
by  amylases,  acids  or  superheated  steam.  The  name 
arises  from  the  fact  that  they  are  strongly  dextrorotatory. 
They  differ  considerably  in  complexity.  There  are  two 
main  varieties  :  erythro-dextrins,  giving  a  reddish  colour 
with  iodine,  and  achroo-dextrins,  which  give  no  colour.  By 
fractionation  with  salt  solutions  Young  has  separated  three 
erythro-dextrins,  I.,  II.,  and  III.  The  first  two  are  precipi- 
tated by  full  saturation  with  ammonium  sulphate,  and  give 
a  purple  and  a  red  colour  respectively  with  iodine.  Erythro- 
dextrin  III.  is  not  precipitated  by  salts,  and  gives  a  red- 
brown  colour  with  iodine.  The  achroo-dextrins  also  are 
not  precipitated  by  salts.  They  are  insoluble  in  strong 
alcohol  and  in  ether.  They  reduce  Fehling's  solution 
slightly,  but  do  not  form  osazones  nor  ferment  with  yeast. 

Stable  dextrin  is  the  dextrin  obtained  from  starch  by 
the  action  of  amylases  continued  until  the  hydrolysis  shows 
an  apparent  equilibrium.  It  is,  as  its  name  implies,  very 
resistant  to  the  further  action  of  the  enzymes,  but  is 
apparently  broken  down  slowly  to  maltose  and  glucose. 


CH.    V.]  STARCH.  119 

It  is  possible  to  regard  it  as  being  formed  by  the  condensa- 
tion of  forty  molecules  of  glucose  with  the  elimination  of 
thirty-nine  molecules  of  water.  On  this  assumption  its 
formula  would  be  39  (C6H10O5)  C6H12O6.  Its  reducing 
power  is  slight,  and  has  [a]D  about  +  195°.  At  the  stage 
of  apparent  equilibrium  in  the  action  of  amylases  on 
starch,  about  85  per  cent,  of  the  initial  weight  is  in  the  form 
of  maltose,  and  about  19  per  cent,  of  stable  dextrin  is 
formed.  The  increase  in  weight  is  due  to  the  addition  of 
the  elements  of  water  in  the  hydrolysis.  The  following 
equation  has  been  suggested  by  Brown  and  Millar. 

100  (C6H10O5)  +  8 1  H2O  =  80  C12H22OU  +  (C6H10O5)39C6H12O6 

Starch.  Maltose.  Stable  dextrin. 

Motto-dextrin  is  the  name  given  to  an  achroo-dextrin 
obtained  by  the  action  of  malt  diastase  in  starch.  It  is 
rapidly  and  completely  hydrolysed  to  maltose  by  the 
amylases. 

The  exact  relationships  between  these  various  dextrins 
to  one  another  and  to  the  constituents  of  the  starch  grain 
are  so  imperfectly  understood  at  present  that  it  is  not 
considered  advisable  to  give  a  scheme  of  the  stages  of 
hydrolysis. 

132.  Place  a  small  amount  of  dry  potato-starch  on  a  slide,  add 
a  drop  of  water,  cover  with  a  slip,  and  examine  under  the  microscope. 
Note,  the  characteristic  oval  starch  grains,  the  concentric  markings 
and  the  hilum,  usually  eccentric.     Make  a  drawing  of  the  grains. 
Run  a  drop  of  iodine  under  the  slip ;  note  that  the  grains  take  on  a 
blue  colour. 

133.  Shake  a  small  amount  of  potato  starch  with  cold  water. 
The  starch  does  not  dissolve.     Filter,  and  add  a  drop  of  iodine 
solution  to  the  nitrate.     The  characteristic  blue  reaction  is  not 
obtained. 

134.  Shake  some  dry  starch  with  a  little  sodium  carbonate 
solution.     No  change  is  effected.     Repeat,   with  a  little  sodium 
hydoxide.     The  starch  is  immediately  gelatinised.     Add  a  few  drops 


120  THE    CARBOHYDRATES.  [cH.    V. 

of  iodine  solution,  a  blue  colour  is  not  obtained.     Treat  with  strong 
acetic  acid.     A  deep  blue  colour  appears. 

NOTE.—  Free  iodine  is  necessary  to  give  the  blue  adsorption  compound 
with  starch.  Sodium  hydroxide  removes  free  iodine,  converting  it  into  iodide 
and  iodate.  The  action  of  the  acid  on  the  latter  causes  the  appearance  of  free 
iodine  and  the  blue  colour.  Always  neutralise  an  alkaline  solution  before  testing 
for  the  polysaccharides.  The  following  equations  show  the  effect  of  sodium 
hydroxide  on  iodine,  and  of  acid  on  a  mixture  of  iodide  and  iodate  : 

(i.)     3l2  +  6NaOH  =  5NaI  +  NaIO3  +  3H2O. 
(ii.)     5NaI  +  NaIO3  +  6HC1  =  3l2  +  6NaCl 


135.  Preparation  of  starch  paste.     Boil    about    75    cc.    of 
distilled  water  in  a  beaker.     Weigh  out  I  gram,  of  dry  potato  starch 
in  another  small  beaker,  add  about  10  cc.  of  cold  water,  and  stir  to 
get  a  uniform  suspension.     Pour  this  into  the  boiling  water  and  stir 
well.     Wash  the  small  beaker  out  with  another  10  cc.  of  cold  water, 
adding  this  to  the  boiling  fluid.     Stir  again,  and  keep  boiling  for  i 
minute.     Cool,  and  make  the  volume  up  to  TOO  cc.     Note  that  the 
"  solution  "  is  distinctly  opalescent.     It  should  be  quite  uniform  and 
free  from  lumps. 

136.  To  a  small  amount  of  the  paste  add  a  drop  or  two  of 
dilute  iodine.     A  deep  blue  colour  is  produced. 

NOTE.  —  The  iodine   solution  should  be  about   o-oi  N.      (See  appendix, 
p.  390.) 

137.  Treat  5  cc.  of  the  cold  starch  paste  with  an  equal  bulk  of 
saturated  ammonium  sulphate.     Shake  the  test-tube  and  allow  it  to 
stand  for  five  minutes.     The  starch  is  precipitated.     Filter  through 
a  dry  paper,  and  add  a  drop  of  iodine  solution  to  the  nitrate.     No 
blue  colour,  or  only  the  very  slightest  tint  is  obtained,  showing  that 
the  whole  of  the  starch  paste  is  precipitated  by  half-saturation  with 
ammonium  sulphate. 

138.  Boil  5  cc.  of  the  starch  paste  with  two  drops  of  concen- 
trated sulphuric  acid  for  about  15  seconds.     Note  that  the  solution 
becomes  perfectly  clear  and  translucent.     Add  two  drops  of  strong 
ammonia  to  neutralise  the  acid,  cool  under  the  tap,  add  an  exactly 
equal  bulk  of  saturated  ammonium  sulphate,  shake  the  tube  vigor- 
ously, and  allow  it  to  stand  for  five  minutes.     Filter  through  a  dry 
filter-paper  and  add  two  drops  of  iodine  solution  to  the  filtrate.     A 
deep  blue  colour  is  obtained. 


CH.    V.j  STARCH.  121 

NOTE. — Starch  paste  is  rapidly  converted  into  "  soluble  starch  "  on  boiling 
with  dilute  mineral  acids.  Soluble  starch  differs  from  starch  paste  in  that  it  is 
not  completely  precipitated  by  half-saturation  with  (NH4)2SO4  in  the  course  of 
five  minutes.  If  it  be  allowed  to  stand  for  twenty-four  hours,  however,  it  is 
completely  precipitated. 

139.  Measure  2  cc.  of  the  paste  into  a  test-tube,  add  6  drops 
of  concentrated  hydrochloric  acid  by  means  of  a  dropping  pipette 
(see  fig.  5).       Heat  to  J  boiling   and  maintain  the  boiling  for  one 
minute   by   the  watch.     Cool  thoroughly  under   the    tap.      Add 
first  one  drop,  and  then  another  drop,  of  the  iodine  solution.     A  red 
or    violet    colour   is   produced,    indicating  the  conversion  of  the 
starch  into  erythro-dextrin  by  acid  hydrolysis. 

140.  Boil  2  cc.  of  the  paste  with  6  drops  of  concentrated 
hydrochloric  acid  as  before.     Cool.     Add  3  drops  of  glycerol  and  8 
drops  of  20  per  cent,  copper  sulphate.     Add  20  per  cent,  sodium 
hydroxide  until  a  grey  precipitate  is  produced.     Now  add  another 
2  cc.  of  the  sodium  hydroxide  and  boil  for  a  minute.    A  slight 
reduction  is  obtained. 

141.  Repeat  the  previous  exercise,  using  20  drops   of   the 
acid,    and   boiling    for  two  minutes.     Cool.      Add   3   drops     of 
glycerol  and  16  drops  of  the  copper  sulphate.     Neutralise  with  soda 
and  then  add  2  cc.  in  excess.      Boil  for  one  minute.      Complete 
reduction  of  the  copper  is  obtained,  indicating  that  the  starch  has 
been  hydrolysed  to  glucose  (see  Ex.  102). 

NOTE. — If  12  drops  of  hydrocloric  acid  be  added  and  the  mixture  boiled 
for  one  minute,  it  will  generally  be  found  that  only  a  yellow  cokmr  is  produced 
with  iodine,  and  that  the  amount  of  glucose  formed  is  not  sufficient  to  reduce 
9  drops  of  copper  sulphate.  At  this  stage  a  considerable  proportion  of  the 
carbohydrate  is  in  the  form  of  achroo-dextrin.  It  is  important  to  note  that 
the  complete  hydrolysis  of  starch  by  acids  is  relatively  slow  compared  to  that 
of  sucrose  and  the  other  disaccharides  (see  Ex.  130).  The  addition  of  a 
couple  of  glass  beads  makes  it  easier  to  obtain  smooth  boilmg  in  the  above 
exercises. 

142.  Shake  a  little  commercial  dextrin  with  some  cold  water. 
An  opalescent  solution  is  formed.     Boil  the  solution.     It  becomes 
perfectly  translucent.     (Distinction  from  glycogen.) 

Use  a  3  per  cent,  solution  of  commercial  dextrin  for  the  following  re- 
actions : — 

143.  To  about  5  cc.  of  the  dextrin  solution  add  iodine  solution, 
drop  by  drop,  noting  the  colour  at  every  addition.     The  colour  is  at 


122  THE   CARBOHYDRATES.  [cH.    V. 

first  almost  a  pure  blue,  but  it  changes  through  a  rich  purple-red  to 
a  red-brown  as  the  iodine  is  added. 

NOTE. — Some  samples  of  commercial  dextrin  contain  a  considerable 
amount  of  soluble  starch. 

144.  Repeat  the  above  experiment,  but  boil  and  then  cool  the 
tube  after  each  addition.     The  colour  disappears  on  boiling,  but 
does  not  reappear  on  cooling  until  several  drops  of  iodine  have  been 
added,  unless  much  soluble  starch  is  present. 

145.  Add  a  drop  or  two  of  the  starch  paste  prepared  in  Ex.  135 
to  about  5  cc.  of  the  dextrin  solution.     To  this  mixture  add  diluted 
iodine  solution,  drop  by  drop.     The  first  additions  produce  a  pure 
blue  colour,  and  it  is  not  till  a  considerable  amount  of  iodine  has 
been  added  that  the  solution  acquires  a  purplish  tint. 

NOTE. — The  affinity  of  starch  for  iodine  is  so  much  greater  than  that  of 
dextrin  that  the  characteristic  colour  reactions  of  erythro-dextrin  are  not 
obtained  until  all  the  starch  has  been  saturated  with  iodine.  Even  then  it  is 
sometimes  difficult  to  detect,  owing  to  the  deep  blue  starch  reaction. 

146.  Treat  5  cc.  of  the  dextrin  solution  with  about  10  drops 
of  the  starch  paste :  to  the  mixture  add  an  equal  bulk  of  saturated 
ammonium  sulphate,  shake  vigorously,  and  allow  to  stand  for  five 
minutes.     The  starch  is  precipitated.     Filter  through  a  dry  paper, 
and  to  a  portion  of  the  filtrate  add  a  drop  or  two  of  iodine  solution. 
The  purple  red  reaction  of  erythro-dextrin  is  obtained. 

147.  Saturate  5  cc.  of  the  dextrin  solution  by  boiling  with  an 
excess  of  finely  powdered  ammonium  sulphate.     Note  the  precipitate 
of  erythro-dextrin  produced.     Cool  under  the  tap  and  filter.     To 
the  filtrate  add  a  drop  of  iodine.     A  red-brown  colour  is  produced. 

NOTE. — This  colour  is  due  to  the  fact  that  erythro-dextrin  III.  is  not 
precipitated  by  ammonium  sulphate.  This  is  the  method  employed  for  the 
identification  of  erythro-dextrin  in  the  presence  of  glycogen,  which  is  com- 
pletely precipitated  by  saturation  with  ammonium  sulphate. 

148.  Boil  a  few  cc.  of  the  dextrin  solution  with  a  small  amount 
of  Fehling's  fluid.     A  well-marked  reduction  is  usually  obtained. 

NOTE. — Commercial  dextrin  is  generally  prepared  by  the  action  of  dilute 
acids  on  starch  (see  Ex.  139),  the  action  being  stopped  as  soon  as  a  portion  fails 
to  give  a  blue  colour  with  iodine,  and  the  products  then  being  precipitated  by 
alcohol.  Such  preparations  contain  some  glucose,  and  often  a  little  soluble 
starch.  At  the  same  time  it  must  be  noted  that  the  achroo-dextrins  have  a 
reducing  action  themselves  even  when  thoroughly  separated  from  the  glucose. 


CH.   V.]  GLYCOGEN.  123 

149.  Take  10  cc.  of  the  dextrin  solution  in  a  small  flask ;  add 
30  cc.  of  strong  (95  per  cent.)  alcohol,  place  the  thumb  over  the 
mouth  of  the  flask  and  shake  vigorously  for  some  seconds.     Note 
that  a  portion  of  the  dextrin  is  precipitated  as  a  gummy  mass  which 
sticks  to  the  sides  of  the  flask. 

Pour  off  the  alcohol,  filter  it  and  label  the  filtrate  A.  Rinse 
the  flask  out  with  a  few  cc.  of  alcohol,  shake  off  as  much  of  this 
alcohol  as  possible,  and  add  10  cc.  of  hot  water.  Shake  this  round 
the  flask  till  the  whole  of  the  gummy  precipitate  dissolves.  Divide 
the  solution  into  three  portions,  B,  C,  and  D.  To  B  add  a  drop  of 
iodine :  a  purple  colour  is  produced.  Boil  C  with  a  little  Fehling's 
solution  :  only  a  slight  reduction  takes  place.  Boil  D  with  two  drops 
of  concentrated  sulphuric  acid  for  two  minutes,  neutralise  with 
sodium  hydroxide,  and  boil  with  a  little  Fehling's  solution :  a  well- 
marked  reduction  occurs. 

150.  To  a  portion  of  filtrate  A,  add  a  drop  of  iodine  solution. 
No  colour  is  produced.     To  another  portion  of  about  5  cc.  add  an 
equal  bulk  of  strong  alcohol.     A  white  precipitate  of  achroo-dextrin 
is  formed. 

Glycogen  is  a  reserve  polysaccharide  found  in  the  liver 
and  muscles.  It  forms  a  white  amorphous  powder, 
soluble  in  water  to  form  an  opalescent  solution.  It  is 
precipitated  from  solution  by  the  addition  of  an  equal 
volume  of  strong  alcohol  or  by  full  saturation  with  am- 
monium sulphate.  It  does  not  reduce  Fehling's  solution, 
form  an  osazone  nor  ferment  with  yeast.  It  gives  a  reddish 
colour  with  iodine.  By  boiling  acids  it  is  hydrolysed  to 
glucose  :  by  most  of  the  diastatic  enzymes  to  maltose,  but 
by  the  diastase  found  in  the  liver  to  glucose.  It  is  not 
affected  by  boiling  alkalies.  It  is  dextro-rotatory. 

Estimation.  Pfliiger's  method  is  undoubtedly  the  best.  20  to  100  grams, 
of  the  tissue  is  cut  into  small  pieces  and  placed  in  an  Erlenmeyer  flask  of  Jena 
glass.  100  cc.  of  60  per  cent,  potash  ("  pure  by  alcohol  " — sp.  gr.  i.  438)  is 
added,  a  reflux  condenser  fitted,  and  the  flask  immersed  for  three  hours  in  a 
boiling  water  bath.  The  alkali  destroys  the  proteins  without  attacking 
the  glycogen. 

After  cooling  200  cc.  of  water  and  800  cc.  of  96  per  cent,  alcohol  are  added, 
and  the  mixture  left  to  stand  over-night.  The  glycogen  is  thus  precipitated 
free  from  protein.  The  supernatant  fluid  is  carefully  decanted  and  filtered. 


of   PHARMACY 


124  THE   CARBOHYDRATES.  [CH.   V. 

The  precipitate  is  washed  with  ten  times  its  volume  of  66  per  cent,  alcohol, 
containing  i  cc.  per  litre  of  saturated  sodium  chloride.  After  settling,  the 
fluid  is  filtered  through  the  original  filter  paper.  This  process  is  repeated  once 
more,  and  then  the  precipitate  is  shaken  with  ten  times  its  volume  of  96  per 
cent,  alcohol  and  filtered  through  the  same  paper.  The  precipitate  is  washed 
with  ether,  dissolved  in  boiling  water  and  the  solution  made  up  to  one  litre. 
200  cc.  of  this  are  treated  with  10  cc.  of  concentrated  HC1  and  heated  in  a 
flask  on  a  boiling  water  bath  for  three  hours,  to  convert  the  glycogen  into 
glucose.  After  cooling,  the  solution  is  neutralised  with  20  per  cent,  potash 
and  filtered  through  a  small  paper  into  a  250  cc.  measuring  flask.  The  wash- 
ings from  the  flask  used  for  inversion  are  filtered  through  the  same  paper  to 
remove  the  last  traces  of  glucose,  and  the  solution  brought  up  to  250  cc.  The 
percentage  of  glucose  in  the  solution  is  determined  by  analysis.  This  multi- 
plied by  -927  gives  the  amount  of  glycogen  in  the  200  cc.  of  the  solution  used 
for  inversion,  and  so  the  percentage  in  the  tissue  can  be  readily  calculated. 

Preparation.  A  rabbit,  which  has  had  a  full  meal  of  carrots  some  five 
or  six  hours  previously,  is  killed  by  decapitation.  The  liver  is  cut  out  as 
quickly  as  possible,  and  the  gall-bladder  removed.  The  liver  is  rapidly 
chopped  into  small  pieces,  a  small  portion  being  reserved  for  Ex.  156,  and  the 
remainder  immediately  thrown  into  boiling  water.  After  about  two  minutes 
boiling  the  larger  morsels  are  strained  off,  pounded  to  a  paste  with  sand  in  a 
mortar,  and  replaced  in  the  boiling  water.  The  proteins  in  solution  are  then 
coagulated  by  making  the  boiling  fluid  just  acid  with  acetic  acid.  The  fluid 
is  filtered  through  coarse  filter  paper.  In  this  way  a  crude  solution  of  glycogen 
is  obtained. 

151.  Boil  5  cc.  in  a  test-tube.     The  characteristic  opalescence 
does  not  disappear.     (Distinction  from  erythro-dextrin.) 

152.  To  a  small  amount  of  the  cooled  solution  add  iodine, 
drop  by  drop.     A  red  colour  is  formed,  which  disappears  on  shaking, 
until  with  a  certain  amount  of  iodine  added  it  is  permanent.     Now 
heat  the  solution.     The  colour  disappears,  to  reappear  on  cooling. 

NOTE. — If  much  protein  is  present  in  solution  the  colour  will  not  re- 
appear on  cooling  unless  a  considerable  amount  of  iodine  be  added.  This  is 
due  to  the  fact  that  proteins  combine  with  iodine  to  form  an  iodo-protein. 

153.  Saturate   10   cc.   of  the   solution  with   finely-powdered 
ammonium  sulphate.     The  glycogen  is  precipitated.     Filter,   and 
add  a  drop  or  two  of  iodine  to  the  nitrate.     No  red  colour  is  pro- 
duced.    Scrape  the  precipitate  off  the  paper,  boil  with  a  small 
amount  of  water.     The  solution  is  markedly  opalescent.     Cool  the 
solution,  and  add  iodine.     A  port-wine  red  colour  is  obtained. 

154.  Boil  5  cc.  of  the  solution  with  a  little  Fehling's  fluid.     A 
very  slight  or  no  reduction  is  obtained. 

NOTE. — If  the  liver  has  been  rapidly  boiled,  no  sugar  will  be  present.  If 
delay  has  occurred  during  the  initial  stages  of  the  preparation,  some  of  the 
glycogen  will  have  been  converted  into  glucose.  (See  Ex.  156.) 


CH.   V.]  QUANTITATIVE    ESTIMATION.  125 

155.  To  10  cc.  of  the  solution  add  20  cc.  of  strong  alcohol, 
shake  vigorously  and  filter.  To  a  portion  of  the  filtrate  add  iodine 
solution.  No  colour  is  obtained,  showing  that  the  whole  of  the 
glycogen  is  precipitated.  Dissolve  the  precipitate  in  a  little  hot 
water :  note  that  it  is  opalescent.  Add  three  drops  of  strong  sulphuric 
acid  and  boil  for  about  three  minutes :  the  opalescence  disappears. 
Neutralise  with  sodium  hydroxide  and  apply  Fehling's  test.  A 
marked  reduction  occurs,  due  to  the  conversion  of  the  glycogen  into 
glucose  by  the  boiling  acid. 


D.    The  Quantitative  Estimation  of  the  Carbohydrates. 

A  very  large  number  of  methods  have  been  introduced 
for  the  estimation  of  glucose,  etc.,  and  considerable  experi- 
ence is  required  to  select  the  method  best  suited  for  a  given 
purpose. 

The  following  scheme  indicates  the  principle  of  the  more 
important  methods,  only  a  few  of  which  are  described 
below  or  in  other  sections  of  this  book. 


A.     Direct  Volumetric  Methods. 

1.  Fehling's.     A   standard   solution   of   copper   sulphate   in 
Rochelle  salt  and  caustic  soda  is  boiled,  and  the  sugar  solution  is 
run  in  from  a  burette  until  the  copper  is  reduced,  as  judged  by  the 
disappearance  of  the  blue  colour.     The  end  point  is  difficult  to  see 
owing  to  the  red  precipitate  that  forms. 

2.  Ling's    modification.    An    external    indicator    containing 
ferrous  thiocyanate  is  employed.     This  is  oxidised  to  red  ferric 
thiocyanate  by  cupric  salts,  but  not  by  cuprous. 

3.  Pavy's.     Strong  ammonia  is  added  to  Fehling's  solution. 
A  measured  amount  of  this  is  boiled  and  the  sugar  solution  run 
in  from  a  burette.  The  cuprous  oxide  formed  is  kept  in  solution  by 
the  ammonia  (see  Ex.  97,  note  5),  forming  a  colourless  compound. 
The  end  point  is  thus  much  easier  to  see.     The  practical  difficulty 

of  the  method  is  to  regulate  the  heating  and  also  the  rate  at  which 


126  THE   CARBOHYDRATES.  [cH.   V. 

the  sugar  solution  is  added.  I  have  abandoned  the  method  for 
routine  class  work,  owing  to  the  considerable  errors  made  by  a 
large  proportion  of  the  students. 

4.  Benedict's.     See  p.   127. 

5.  Folin  and  McEllroy's.     See  p.  129. 

B.      Indirect  Volumetric  Methods. 

6.  Amos  Peters'.     See  p.  134. 

7.  Bertrand's.     The  sugar  is  heated  with  an  excess  of  Fehling's 
solution.     The  cuprous  oxide  formed  is  filtered  off  through  asbestos 
and  dissolved  in  an  acid  solution  of  ferric  sulphate,  some  of  which  is 
reduced  to  ferrous  sulphate.     The  amount  of  the  latter  is  determined 
by  titration  with  standard  permanganate.     By  reference  to  tables 
or  a  curve  the  amount  of  sugar  present  is  readily  calculated. 

8.  Wood-Ost's.     Seep.  131.     This  is  very  similar  to  the  above, 
except  that  a  solution  of  copper  bicarbonate  is  used  instead  of 
Fehling's.     The  amount  of  copper  reduced  by  a  given  weight  of 
sugar  is  thus  nearly  doubled,  and,  owing  to  the  reduced  alkalinity, 
other  substances  do  not  so  readily  affect  the  results. 

9.  Bang's.    See  p.  253,  Ex.  312,  note  2.     An  alkaline  solution 
of  copper  in  potassium  chloride  is  boiled  with  the  sugar  and  a 
measured  amount  of  potassium  iodate.      On  acidifying  the  solution 
with  sulphuric,  the  iodic  acid  oxidises  the  cuprous  oxide  to  cupric 
oxide.     The  loss  of  iodic  acid  is  determined  by  adding  potassium 
iodide  and  titrating  with  thiocyanate.  The  method  has  been  adapted 
for  the  estimation  of  sugar  in  very  small  quantities  of  blood. 

C.    Colorimetric  Methods. 

10.  Benedict's.    See  p.   251.    The  sugar  solution  is   heated 
with  sodium  carbonate  and  sodium  picrate.     The  picrate  is  reduced 
to  picramate,  which  is  estimated  colorimetrically  against  picramic 
acid,,  either  a  solution  of  the  pure  substance,  or  one  prepared  from  a 
standard  solution  of  glucose. 


CH.  v.]  BENEDICT'S  METHOD.  127 

D.    Polarimetric  Method. 

ii.  This  is  of  great  value.  The  relationships  between  reduc- 
ing and  rotatory  powers  of  solutions  before  and  after  hydrolysis 
must  be  determined  for  the  identification  and  analysis  of  mixed 
carbohydrates. 

Of  the  various  methods  proposed,  the  Author  is  of  the  opinion  that  for 
ordinary  routine  work  and  for  urinary  analysis,  Benedict's  direct  volumetric 
method  is  the  most  reliable  in  the  hands  of  the  majority  of  workers.  The 
recent  method  of  Folin  and  McEllroy  has  certain  advantages,  especially  in  the 
cost  of  materials,  and  may  eventually  supersede  Benedict's.  The  Wood-Ost 
process  is  worthy  of  more  extended  recognition.  In  spite  of  the  10  minutes' 
boiling  that  is  necessary,  the  method  is  a  rapid  one,  and  when  completed  one  is 
left  with  a  sense  of  confidence  that  is  somewhat  lacking  in  the  direct  methods. 

For  accurate  research  work  the  method  of  Amos  Peters  is  extremely 
satisfactory. 

When  a  large  series  of  diabetic  urines  have  to  be  examined  sufficiently 
approximate  results  can  be  obtained  by  polarisation,  after  removal  of  the 
pigment  by  blood  charcoal  in  the  presence  of  10  per  cent,  of  acetic  acid,  a  little 
freshly  prepared  metaphosphoric  acid  being  added  if  proteins  are  present. 

158.    Benedict's  Method. 

Principle  of  the  Method. — An  alkaline  solution  of  copper  sul- 
phate, containing  thiocyanate  is  boiled  and  the  sugar  solution  run 
in  from  a  burette  till  the  blue  colour  just  disappears.  The  thio- 
cyanate forms  a  white  insoluble  compound  with  the  cuprous  hydroxide 
formed  by  the  reduction  of  the  copper,  and  so  there  is  no  red  cuprous 
oxide  precipitated  to  obscure  the  blue  tint.  A  little  potassium 
ferrocyanide  is  also  added  to  prevent  any  possibility  of  the  deposi- 
tion of  the  cuprous  oxide. 

Preparation  of  the  Solution. — With  the  aid  of  heat  dissolve 
Sodium  citrate  . .         . ..         . .         200  grams. 

Sodium  carbonate  (cryst.)  . .         200  grams. 

(or  anyhdrous  sod.  carb.  75  grams.) 
Potassium  thiocyanate  (sulphocyanide)       125  grams. 

in  enough  distilled  water  to  make  about  800  cc.  of  the  mixture  and 
filter,  and  cool  to  room  temperature. 

Dissolve  1 8  grams,  of  the  purest,  air-dried  crystalline  copper 
sulphate  in  about  100  cc.  of  distilled  water,  and  pour  it  slowly  into 
the  other  liquid  with  constant  stirring.  Add  5  cc.  of  a  5  per  cent, 
solution  of  potassium  ferrocyanide  and  then  distilled  water  to  make 


128 


THE   CARBOHYDRATES. 


[CH.   V. 


the  total  volume  1000  cc.    The  solution  appears  to  keep  indefinitely, 
without  any  special  precaution,  such  as  exclusion  of  light,  etc. 

Method  of  Analysis. — Fit  a  150  cc.  flask 
into  a  ring  of  a  retort  sf  and  of  such  a  si/e  that 
it  is  fairly  firmly  held.  There  is  no  need  to 
use  a  wire  gauze.  Arrange  the  flask  at  such  a 
height  over  a  Bunsen  burner  that  the  reagent 
can  be  kept  briskly  boiling  by  means  of  a 
small  flame.  In  the  flask  place  3  to  4  grams, 
of  anhydrous  sodium  carbonate.  This  can  be 
roughly  measured  by  taking  a  depth  of  I  inch 
in  a  dry  test-tube.  Then  add  25  cc.  of  the 
reagent  and  heat  till  most  of  the  carbonate  is 
in  solution.  Run  the  sugar  solution  in  from 
a  burette,  which  is  best  held  in  the  hand. 
Run  the  sugar  in  slowly,  till  a  bulky  chalk- 
white  precipitate  is  formed  and  the  blue  colour 
lessens  perceptibly  in  intensity.  From  this 
point  the  sugar  is  added  more  and  more 
slowly,  with  constant  boiling,  until  the  dis- 
appearance of  the  last  trace  of  blue  colour, 
which  marks  the  end-point.  If  the  volume  of 
the  sugar  is  less  than  5  cc.,  dilute  it  accurately 
with  water  till  about  idee,  are  judged  necessary. 
Repeat  the  titration  with  this  as  before. 

NOTES. — There  is  a  tendency  to  run  in  an  excess  of  the  sugar,  unless 
special  care  is  exercised  throughout  the  titration,  and  particularly  at  the  end. 
The  solution  must  be  kept  steadily  boiling  during  the  entire  process,  and 
towards  the  end  the  sugar  must  be  added  in  portions  of  a  drop  or  two,  with  an 
interval  of  about  30  seconds  after  each  addition.  Should  the  mixture  become 
too  concentrated,  boiled  distilled  water  may  be  added  to  replace  that  lost  by 
evaporation. 

The  titration  can  also  be  carried  out  in  a  white  porcelain  dish  of  10  to 
15  cm.  in  diameter,  but  the  risk  of  reoxidation  of  the  cuprous  compound  is 
greater  than  in  a  flask. 

Should  the  solution  bump  excessively,  two  or  three  small  pieces  of  broken 
porcelain  may  be  added. 

The  3  to  4  grams,  of  anhydrous  sodium  carbonate  are  added  to  produce  the 
necessary  alkalinity.  This  proportion  of  alkali  cannot  be  added  to  the  bulk 
of  the  standard  solution,  for  it  would  crystallise  out  at  room  temperature. 


Fig.  1 3  A. 

Apparatus  for 
Benedict's  Method. 


CH.    V.]  METHOD    OF    FOLTN    AND    McELLROY.  129 

Calculation  of  Results. 

25  cc.  of  Benedict's  solution  are  reduced  by  0-05      gram,  of  glucose. 

0-053    gram,  fructose. 
0-074    gram,  maltose. 
0-0676  gram,  lactose. 
Example. — First  titration  required  2-4  cc. 

Solution  diluted  i  in  4  (10  cc.  of  sugar  diluted  with  30  cc.  water). 

Second  titration  required  9-7  cc. 

So  9-7  cc.  diluted  solution  contain  0-05  gram,  glucose. 

10  cc.  diluted  solution   contain   '°5  x  IO° 

9-7 

,     .        O-O5    X    TOO    X    4 

100  cc.  original  solution  contain 

9-7 

Percentage  of  glucose  =  2-06. 

159.    The  method  of  Folin  and  McEllroy.* 

Principle.  Five  cc.  of  a  6  per  cent,  solution  of  crystalline 
copper  sulphate  are  treated  in  a  large  test-tube  with  a  mixture  of 
sodium  phosphate,  sodium  carbonate  and  sodium  or  potassium 
thiocyanate  in  the  solid  form.  A  clear  blue  solution  is  obtained  on 
boiling.  The  sugar  solution  is  run  in  from  a  burette.  A  white 
precipitate  of  cuprous  thiocyanate  is  formed.  The  sugar  is  run  in 
very  slowly  until  the  blue  copper  colour  is  just  discharged.  Owing 
to  the  reduction  of  the  alkalinity  of  the  solution,  the  amount  of 
copper  reduced  by  a  given  amount  of  sugar  is  considerably  greater 
than  in  Fehling's  or  Benedict's  methods.  Also  the  reoxidation 
of  the  cuprous  salts  to  the  cupric  condition  is  relatively  very  slow. 
The  main  objection  to  the  method  is  the  relatively  slow  rate  of 
reduction. 

Reagents  required  : 

1.  Copper  sulphate.     Dissolve  60  grams,  of  the  best  air-dried  crystalline 
copper  sulphate  in  distilled  water,  add  2  or  3  cc.  of  pure  sulphuric  acid,  and 
make  the  volume  up  to  i  litre.     The  acid  is  to  prevent  the  slow  deposition  of 
copper  hydroxide  and  silicate  due  to  the  action  on  the  glass. 

2.  A  Ikaline  phosphate  powder.     Mix  together  in  a  large  mortar,  100  grams, 
of   disodium  phosphate  crystals   (HNa2PO4i2H2O),   60  grams,  of  anhydrous 
sodium  carbonate  and  30  grams,  of  sodium  or  potassium  thiocyanate.    Accord- 
ing to  the  authors  this  mixture  keeps  indefinitely,  but  in  England  it  is  apt 
to  absorb  moisture  and  resolve  itself  into  a  somewhat  pasty  mass. 

*  Journ.  of  Biological  Chemistry,  xxxiii.,  p.  516  (1918). 


130 


THE   CARBOHYDRATES. 


[CH.    V. 


3.  Special  Burette.  Folin  and  McEllroy  describe  an  ingenious  method  of 
measuring  small  quantities  of  fluids  by  means  of  an  accessory  fine  tip  fitted  to  an 
ordinary  burette.  When  the  fluid  is  dropping  from  the  burette  very  slowly, 
the  size  of  the  drop  is  constant  for  a  particular  fluid.  So  if 
the  number  of  drops  emitted  by  a  given  tip  for  a  delivery  of 
i  cc.  be  known,  the  volume  of  the  number  of  drops  required 
for  the  titration  can  be  readily  calculated.  The  author  pre- 
fers to  use  the  special  5  cc.  burette  shewn  in  fig.  14.  It  is 
provided  with  an  accessory  tip,  which  is  drawn  out  to  a  fine 
point.  As  recommended  by  Folin  and  McEllroy,  the  burette 
must  be  filled  by  suction.  Elaborate  washing  of  the  burette 
in  the  intervals  between  successive  estimations  is  thereby 
rendered  unnecessary. 

Method.  Weigh  out  approximately  5  grams,  of 
the  phosphate  mixture  and  transfer  it  to  a  clean,  dry 
tube,  conveniently  7  inches  by  f  ths  inch.  Add  5  cc. 
of  the  6  per  cent,  solution  of  copper  sulphate,  shake 
and  heat  to  boiling.  A  deep  blue,  nearly  clear, 
solution  is  obtained.  It  is  advisable  to  improvise 
a  test-tube  holder  by  folding  a  piece  of  paper.  Run 
in  about  0-5  cc.  of  the  sugar  solution  from  the  burette 
and  boil  very  gently  for  2  minutes  by  the  watch.  The 
tube  should  be  held  at  an  angle  and  moved  about  in 
a  small  flame :  excessive  concentration  and  loss  by 
spurting  can  be  thus  avoided.  If  there  is  an  appre- 
ciable amount  of  sugar  present  a  chalky  white 
precipitate  appears.  If  the  blue  colour  entirely 
disappears  there  is  more  than  5  per  cent,  of  sugar 
present,  and  the  estimation  must  be  repeated  with 
a  diluted  solution.  If  the  copper  is  only  slightly 
reduced,  yielding  only  a  small  amount  of  cuprous 
thiocyanate,  add  a  further  amount  of  the  sugar 
solution  and  boil  gently  for  another  minute.  If  now 
the  greater  part  of  the  copper  has  been  reduced, 
complete  the  titration  by  adding  a  drop  at  a  time, 
boiling  for  i  minute  after  each  addition.  The  total 
period  of  boiling  must  not  [be  less  than  4  minutes, 
and  should  not  exceed  8.  The  copper  value  has  been 
adjusted  to  a  period  of  5  to  6  minutes. 


Fig.  14- 

Burette  with 
accessory  tip. 


A  second  estimation  is  often  necessary.     With  a  little  experi- 
ence it  is  easy  to  judge  of  the  amount  that  should  be  added,  so  that 


CH.   V.]  WOOD-OST   METHOD.  131 

after  a  preliminary  boiling  period  of  3  minutes,  only  a  few  drops  more 
are  required  to  complete  the  tit  ration. 

With  pure  glucose  solutions  the  end  point  is  very  sharp.  With 
lactose,  maltose  and  diabetic  urines  the  end  point  is  the  transition 
from  a  green  to  a  yellow  colour. 

The  special  precaution  necessary  is  to  ensure  that  the  boiling 
period  is  within  the  stated  limits. 

Calculation  of  results. 

5  cc.  of  the  copper  are  reduced  by  25  mg.  glucose. 
,,  ,,  „  25  mg.  fructose. 

„  „  „  40-4  mg.  anhydrous  lactose. 

„  ,,  ,,  45  mg.  anhydrous  maltose. 

In  the  case  of  glucose  the  concentration  in  grams,  per  cent,  is  2-5  divided 
by  volume  of  solution  required. 

160.    The  Wood-Ost  copper  carbonate  method.* 

Principle.  A  solution  of  copper  sulphate  in  carbonate  and 
bicarbonate  of  potash  is  boiled  with  a  given  volume  of  the  sugar 
solution.  The  cuprous  oxide  formed  is  filtered  off  through  asbestos 
and  washed  with  cold  water.  It  is  suspended  in  acid  ferric  sulphate 
and  the  amount  of  ferrous  sulphate  formed  determined  by  titration 
with  standard  potassium  permanganate.  The  amount  of  copper 
reduced  being  known,  the  weight  of  sugar  in  the  volume  taken  can 
be  determined  by  reference  to  a  curve  or  tables. 

Preparation  of  solutions. 

1.  Copper  carbonate.     Dissolve  250  grams,  of  potassium  carbonate  and 
100  grams,  of  potassium  bicarbonate  by  the  addition  of  about  600  cc.  of  warm 
distilled  water.     Dissolve  23*5  grams,  of  pure  crystalline  copper  sulphate  in 
about  200  cc.  of  water.     Gradually  add  the  copper  to  the  carbonate  solution, 
mixing  well  during  the  addition.     Make  the  volume  up  to  1000  cc.  and  filter. 
The  solution  seems  to  keep  indefinitely. 

2.  Acid  ferric  sulphate.     Gradually  add  250  cc.  of  pure  concentrated 
sulphuric  acid  to  750  cc.  of  distilled  water.     Add  25  grams,  of  ferric  sulphate. 
Warm  till  the  sulphate  has  dissolved,  and  filter  if  necessary.     The  solution 
must  not  be  used  until  it  has  cooled. 

3.  Standard  potassium  permanganate.    Dissolve  about  6  grams,  of  potas- 
sium permanganate  in  a  1 100  cc.  of  cold  distilled  water.     Having  made  certain 
that  the  whole  has  dissolved  standardise  the  solution  as  follows  : — Weigh  out 
between  0-3  and  0-4  gram,  of  pure  crystalline  ammonium  oxalate,  determining 


*  T.  B.  Wood   and   A.    Berry,   Cambridge  Philosophical  Journal,  xlvi., 
p.  103  (1904). 


132  THE   CARBOHYDRATES.  [CH.   V. 

the  exact  weight  to  a  milligramme.  Add  about  50  cc.  of  distilled  water,  to 
which  about  3  cc.  of  pure  concentrated  sulphuric  acid  have  just  previously 
been  added.  Warm  on  a  water  bath  until  the  solid  has  completely  dissolved. 
Titrate  the  warm  solution  with  the  permanganate.  This  must  be  run  in  very 
slowly  at  first,  further  additions  not  being  made  until  the  colour  has  com- 
pletely faded.  The  end  point  is  reached  when  a  faint  rose  colour  persists  for  at 
least  a  minute.  If  A  be  the  weight  of  ammonium  oxalate  taken,  and  P  the 
volume  of  permanganate  required,  then  i  cc.  of  permanganate  corresponds  to 

895^x  A  =  T  mg.  copper. 

It  is  convenient  to  have  T  =  10.  If  T  be  greater  than  10,  add  100 
(T  -  io)cc.  of  water  to  i  litre  of  the  solution.  If  the  titration  has  been  con- 
ducted accurately,  i  cc.  of  the  permanganate  corresponds  to  i  mg.  copper. 

The  calculation  is  based  on  the  following  equations  :  — 

Cu2O  +  Fe2(SO4)3  +  H2SO4  =  2  CuSO4  +  2  FeSO4  +  H2O. 

10  FeSO4  +  2  KMnO4  +  8  H2SO4  =  5  Feg(SO4)a  +  ICjSC^  +  2  MnSO4  +  8  H2O. 

5  C2H2O4  +  2  KMnO4  +  3  H2SO4  =  KjSC^  +  2  MnSO4  +  8  H2O  +  10  CO2. 


So  i  mol.  of  oxalic  acid  or  of  ammonium  oxalate  (C2O4N2H8.H2O)  requires  the 
same  amount  of  permanganate  as  2  Fe,  which  corresponds  to  2  Cu. 

2  x  63-6  x  A 

So  P  cc.  of  permanganate  =  —  -  =  0-8951  x  A  gm.  Cu. 

1  42*  i 

Method.  Measure  50  cc.  of  the  copper  solution  into  a  150  cc. 
flask  of  "  Duro  "  glass.  Add  two  or  three  small  pieces  of  broken  por- 
celain to  prevent  subsequent  bumping.  Boil  by  heating  on  a  gauze 
with  a  Bunsen.  As  soon  as  the  solution  has  commenced  to  boil  run 
in  exactly  10  cc.  of  the  sugar  solution,  which  should  be  between 
0-2  and  0-9  per  cent,  of  glucose  (see  note  i).  Note  the  exact  time 
when  the  boiling  recommences.  The  flame  should  be  moderately 
high  at  first,  but  as  soon  as  the  solution  recommences  boiling  after 
the  addition  of  the  glucose,  it  should  be  lowered  so  that  it  just  main- 
tains gentle  boiling.  After  exactly  ten  minutes'  boiling,  stop  the 
reduction  by  immersing  the  flask  .in  cold  water.  Then  cool 
thoroughly  under  the  tap. 

Filtration  of  the  cuprous  oxide.  This  is  done  through  asbestos 
by  means  of  a  Gooch  crucible  of  25  to  50  cc.  capacity  (see  fig.  33),  or, 
better,  through  an  asbestos  mat  supported  on  a  small  (15  mm. 
diam.)  perforated  porcelain  plate,  resting  in  a  conical  funnel  that 
passes  through  a  rubber  stopper  fitting  the  neck  of  a  filtering  flask. 
The  preparation  of  the  mat  and  the  subsequent  filtration  is  much 
facilitated  by  use  of  the  special  pump  connexions  shewn  in  fig.  9,  p.  74. 
The  mat  is  prepared  as  follows  :  the  suspension  of  well-washed 


CH.    V-] 


WOOD-OST    METHOD. 


133 


asbestos  (see  note  2)  is  poured  on  to  the  plate  (or  into  the  Gooch 
crucible)  and  allowed  to  settle  down  without  suction.  After  a  short 
time  the  tube  E  (fig.  9)  is  connected  to  the  filtering  flask,  the  tap  C 
being  turned  so  that  it  connects  to  B  (thus  practically  preventing 
suction  through  E),  and  the  water  pressure  turned  on.  The  tap  C 
is  then  turned  through  an  angle  so  as  to  allow  suction  through  E, 
but  before  the  water  has  been  completely  drained  off,  the  tap  is 
rapidly  opened  again,  it  being  important  not  to  suck  too  hard.  The 
amount  of  asbestos  required  is  such  as  to  form  a  mat  about  2  mm. 
in  depth.  A  small  porous  plate  may  be  placed  on  the  top  of  the  mat 
to  prevent  the  latter  from  being  disturbed  too  much  by  pouring  on 
water  or  the  copper  solution.  The  mat  is  then  washed  two  or  three 
times  with  distilled  water,  gentle  suction  being  applied  after  each 
addition.  The  final  suction  should  be  sufficient  to  make  the  mat 
quite  firm.  The  mat  should  be  prepared  during  the  heating  process, 
it  only  requiring  a  few  minutes,  in  spite  of  this  long  description. 
The  cold  copper  solution  is  poured  on  to  the  upper  porcelain  plate 
and  suction  then  started.  The  pressure  must  be  released  before  all 
the  solution  has  passed  through,  it  being  most  important  to  avoid 
caking  the  cuprous  oxide  by  too  high  a  pressure.  The  flask  is 
washed  out  with  about  10  cc.  of  cold  distilled  water  (which  may, 
with  advantage,  have  been  recently  boiled  and  cooled),  and  this 
poured  on  to  the  cuprous  oxide  on  the  asbestos  and  filtered  through 
as  before.  This  washing  is  repeated  twice  more,  care  being  taken 
all  the  time  to  prevent  caking  of  the  cuprous  oxide  by  too  high  a 
nitration  pressure. 

Solution  of  the  precipitate.  Measure  25  cc.  of  the  ferric  sulphate 
solution  by  means  of  a  measuring  cylinder.  Pour  about  5  cc.  of  this 
into  the  boiling  flask  and  shake  this  round  to  dissolve  any  cuprous 
oxide  that  is  sticking  to  the  walls  of  the  vessel.  Transfer  the  filter- 
ing mat  and  the  filtering  discs  to  a  small  beaker  by  means  of  a  small 
glass  rod  that  has  a  pointed  hook.  Remove  the  funnel  from  the 
filtering  flask  and  wash  it  down  into  the  beaker  with  the  remainder 
of  the  acid  ferric  sulphate.  Wash  out  the  flask  two  or  three  times 
with  small  quantities  of  water,transf erring  this  to  the  beaker  through 
the  funnel.  Wash  down  the  small  rod  and  stir  well  with  a  larger 
glass  rod,  which  should  not  be  guarded  with  a  rubber  collar,  since 
permanganate  attacks  rubber.  The  cuprous  oxide  may  all  dis- 


134  THE   CARBOHYDRATES.  [CH.    V. 

solve,  but  a  certain  amount  may  remain  in  suspension  until  the 
permanganate  titration  is  nearing  completion. 

Titration  of  the  reduced  iron.  Run  in  the  potassium  permanga- 
nate from  a  burette  fitted  with  a  glass  stopcock,  stirring  the  mixture 
well.  From  time  to  time  examine  the  beaker  by  holding  it  above 
the  head.  Any  lumps  of  undissolved  cuprous  oxide  can  thus  be 
detected.  They  must  be  broken  up  and  brought  into  solution  by 
rubbing  with  the  rod.  It  is  most  important  that  this  should  be  done 
before  the  titration  is  completed.  The  end  point  is  reached  when  a 
faint  pink  tinge  persists  for  at  least  ten  seconds. 

Calculation  of  results.  One  cc.  of  permanganate  =  10  mg.  Cu. 
The  amount  of  sugar  corresponding  to  various  amounts  of  copper  are 
obtained  by  plotting  the  results  given  below.  The  amount  of  sugar 
corresponding  to  the  exact  amount  of  copper  reduced  is  thus  found. 
The  number  of  milligrammes  of  sugar  in  10  cc.  divided  by  10  and 
multiplied  by  the  dilution  employed  gives  the  percentage  of  sugar. 

mg.Cu.  mg.Glucose  mg.Maltose     mg.Cu.  mg.Glucose  mg. Maltose 
anhydride     anhydride  anhydride    anhydride 

25  7'3 

50  15 

75  22-4 

100  30 

125  37-8 

150  45*3 

NOTES. — i.  A  rough  approximation  of  the  concentration  of  glucose  in 
the  original  solution  can  be  made  by  use  of  Fehling's  solution.  The  sugar 
should  be  so  diluted  that  3  to  5  cc.  reduce  3  cc.  of  Fehling's  solution. 

2.     Preparation  of  the  asbestos.     (See  appendix.) 

161.  The  estimation  of  glucose  by  the  method  of  Amos 
Peters. 

Principle.  A  known  volume  of  the  sugar  solution  is 
boiled  with  a  measured  amount  of  an  alkaline  solution  of 
copper  sulphate.  The  cuprous  oxide  is  filtered  off  and  the 
copper  in  the  filtrate  determined  by  treatment  with 
potassium  iodide  and  titration  of  the  iodine  liberated  by 


14-0 

175 

53 

99 

28-2 

2OO 

60-5 

H3 

42-3 

225 

69-5 

130-4 

56-2 

250 

79'2 

147-5 

70*5 

275 

89 

164-6 

84*5 

290 

95'4 

175 

CH.   V.] 


METHOD   OF   AMOS    PETERS. 


135 


means  of  a  solution  of  sodium  thiosulphate.  From  the 
amount  of  copper  reduced  the  amount  of  glucose  in  the 
volume  of  solution  taken  can  be  determined. 

Solutions  required. 

1.  Copper  sulphate.     69-278  grams,  of  the  purest  crystalline  salt  CuSO4, 
5H2O,  is  dissolved  in  water  and  the  volume  made  up  to  i  litre. 

2.  Alkaline  tartrate.     346  grams,  of  Rochelle  salt  and  250  grams,  of  pure 
potassium  hydroxide  are  dissolved  in  water  and  the  volume  made  up  to  i  litre. 

3.  Sodium  thiosulphate.     99-2    grams,  of  the  purest  thiosulphate  are 
dissolved  in  boiled  out  distilled  water  and  the  volume  made  up  to  i  litre  with 
boiled  out  distilled  water.     It  should  be  prepared  at  least  a  week  before  it  is 
standardised. 

4.  Potassium  iodide.     Saturated  solution.     100  grams,  of  the  solid  are 
treated  with  70  cc.  of  hot  distilled  water  and  the  solution  allowed  'to  cool. 

5.  Soluble  starch.  Shake  i  gram,  of  soluble  starch  (see  p.  391)  with  about 
10  cc.  of  distilled  water  and  pour  the  suspension  into  90  cc.  of  boiling  water. 

Standardisation  of  the  thiosulphate.  Measure  20  cc.  of 
the  copper  sulphate  into  a  200  cc.  Erlenmeyer  flask.  Add  40  cc. 
of  distilled  water  and  20  cc.  of  strong  (33  per  cent.)  acetic  acid. 
Insert  a  thermometer  and  cool  or  warm  to  20°  C.  Run  in  about 
6-5  cc.  of  the  saturated  potassium  iodide,  the  thermometer  being 
withdrawn  and  its  stem  washed  with  this  solution.  The  iodine 
liberated  is  titrated  at  once  with  the  thiosulphate.  When  approach- 
ing the  end  point  add  about  i  cc.  of  the  soluble  starch.  The  colour 
changes  to  a  chocolate  brown  when  very  near  the  end  point.  This  is 
best  determined  by  the  "  spot  test  "  method.  Allow  a  drop  of  the 
thiosulphate  to  fall  on  the  quiet  surface  of  the  liquid.  If  the  end 
point  has  not  been  reached,  a  very  perceptible  white  area  is  seen 
around  the  drop.  This  is  very  readily  distinguished  from  the 
diminution  of  the  slightly  yellowish  colour  of  the  suspended  cuprous 
iodide.  The  volume  of  the  drop  delivered  by  the  burette  must  be 
deducted  from  the  total  volume  added. 

The  copper  value  of  the  thiosulphate  is  calculated  as  shewn  in 
the  following  example  :  — 

20  cc.  of  the  copper  sulphate  =  352-93  mg.  Cu. 
This  required  27-6  cc.  of  thiosulphate. 


So  i  cc.  of  thiosulphate  = 


27-6 


=  1278  mg.  Cu. 


136 


THE   CARBOHYDRATES. 


[CH.   V. 


The  heating  apparatus.  -Use  the  apparatus  shewn  in  fig.  15. 
In  a  200  cc.  Erlenmeyer  flask  of  Resistance  glass,  and  of  about  6  cm. 
basal  diameter,  place  60  cc.  of  distilled  water.  The  flask  is  fitted 
with  a  2-hole  rubber  stopper  carrying  a  thermometer  so  graduated 
that  the  stem  above  34°  C.  is  visible  above  the  upper  edge  of  the 
stopper.  The  lower  end  of  the  thermometer  should  be  about  2  mm. 
from  the  bottom  of  the  flask. 


Fig.  15.  Cole's  apparatus  for  maintaining  a  standard  heating  power. 
The  manometer  tube  contains  a  dilute  solution  of  eosin  or  other  dye.  It 
also  contains  a  globule  of  mercury  which  nearly  fills  the  bottom  of  the  tube. 
This  prevents  the  rapid  oscillations  of  pressure  due  apparently  to  the  explo- 
sions of  local  gas  engines. 

Turn  on  the  tap  B  to  its  full  extent  and  light  the  flame  of 
a  Bunsen  or  Meker  burner,  which  is  placed  under  a  piece  of  asbestos 
gauze  carried  by  an  adjustable  ring  stand.  The  gauze  should  be 
from  4  to  6  cm.  above  the  top  of  the  burner.  Tighten  the  screw  A 
till  the  pressure  is  reduced  about  one-third.  Allow  the  gauze  to  get 
thoroughly  heated  and  then  place  the  flask  in  the  centre  of  the 


CH.    V.] 


METHOD    OF   AMOS    PETERS. 


137 


heated  gauze.  By  means  of  a  stop-watch  note  the  time  for  the 
temperature  to  rise  from  35°  to  95°.  If  the  time  is  greater  or  less 
than  120  sees,  loosen  or  tighten  the  screw  A  and  repeat  the  experi- 
ment with  another  60  cc.  of  distilled  water  until  the  temperature  of 
the  water  rises  from  35°  to  95°  in  120  ±  2  sees.  The  height  of  the 
ring  and  the  thickness  of  the  asbestos  should  be  such  that  the  pressure 
is  well  under  the  minimum  supplied  to  the  laboratory  and  yet 
sufficient  to  prevent  any  risk  of  the  flame  striking  back.  Note  the 
manometer  reading.  The  standard  heating  power  can  be  rapidly 
obtained  for  further  experiments  by  adjusting  the  screw  A  so  that 
the  manometer  shews  the  requisite  pressure. 

Filtering  Apparatus.       It  is  convenient  to  use  the  apparatus 
shown  in  Fig.  16.    A  is  a  "  Duro  "  flask  of  200  cc.  capacity.    Tube  B 

is  an  ordinary  calcium  chloride 
tube.  The  lower  end  should 
reach  at  least  3  cm.  below  the 
lower  edge  of  the  stopper  to 
prevent  loss  by  splashing  during 
filtration.  The  filtering  mat  is 
made  of  glass  wool,  asbestos 
fibre,  powdered  pumice  and 
asbestos  fibre  added  in  that 
order.  The  mat  should  be 
washed  with  nitric  acid  and  then 
thoroughly  washed  with  water. 
After  a  test  the  cuprous  oxide 
on  the  mat  is  dissolved  in  nitric 
acid  diluted  with  an  equal 
volume  of  water  and  then 
thoroughly  washed. 

An  ordinary  Gooch  crucible 
can  be  used  with  a  mat  pre- 
pared in  the  same  way.      The 
arrangement  is  shewn  in  fig.  33,  p.  259. 


Fig.  1 6.     Filtering  apparatus  for 
reduced  copper. 


Method  of  Analysis.  Into  a  200  cc.  Erlenmeyer  flask  measure 
20  cc.  of  the  standard  copper  sulphate,  20  cc.  of  the  alkaline 
tartrate,  and  20  cc.  of  the  sugar  solution  (which  must  not  contain 


138 


THE   CARBOHYDRATES. 


[CH.    V. 


CH.   V.]  METHOD   OF   AMOS   PETERS.  139 

more  than  180  mg.  of  glucose).  Fit  the  two-holed  rubber  stopper 
firmly  into  the  neck  of  the  flask,  adjust  the  thermometer  so  that 
its  lower  end  is  2  mm.  from  the  bottom  of  the  flask  and  place  on 
the  heated  gauze.  Note  the  time  when  the  mercury  indicates  a 
temperature  of  95°  C.  Allow  the  heating  to  continue  for  exactly 
20  sees,  beyond  this.  Remove  the  flask  by  gripping  the  rubber 
stopper  and  swill  it  for  a  second  or  two  under  the  tap  or  in  a  bowl  of 
water.  The  reduction  of  the  temperature  practically  stops  the 
reduction.  Filter  the  hot  fluid  at  once,  using  the  stem  of  the  ther- 
mometer as  a  stirring  rod.  Wash  the  flask  twice  with  about  7  cc. 
of  distilled  water.  Cool  the  filtrate  by  holding  the  flask  under  the 
tap.  Add  exactly  4  cc.  of  strong  sulphuric  acid,  insert  a  ther- 
mometer and  cool  to  20°.  Add  6-5  to  7  cc.  of  the  saturated  solution 
of  potassium  iodide,  washing  the  stem  of  the  thermometer  with  this 
solution.  Titrate  at  once  with  the  standardised  solution  of  sodium 
thiosulphate  as  described  above,  using  soluble  starch  as  an  indicator 
when  near  the  end  point. 

Calculation  of  results.  From  the  amount  of  thiosulphate 
required  the  amount  of  copper  in  the  filtrate  is  determined.  Know- 
ing the  amount  taken  (352-9  mg.),  the  amount  reduced  by  the  sugar 
can  be  calculated.  The  amount  of  glucose  corresponding  to  this 
copper  can  be  determined  by  a  reference  to  the  curve  in  Fig.  j  7. 

Example.  The  copper  in  the  filtrate  required  14-62  cc.  of 
thiosulphate. 

i  cc.  of  thiosulphate  =  12-86  mg.  Cu. 

So  copper  in  filtrate  =  14-62  x  12-86  =  188-0  mg.  Cu. 

So  copper  reduced  by  glucose  in  20  cc.= 352 -9— 188-0=164-9  mg. 

From  the  curve  this  is  seen  to  correspond  to  86-3  mg.  glucose. 

So  20  cc.  contain  86-3  mg.  glucose. 

So  100  cc.  contain  431-5  mg.  glucose.     =  0-431  per  cent. 

NOTE. — If  the  amount  of  reduced  copper  is  between  60  and  200  mg.,  the 
amount  of  glucose  corresponding  to  this  can  be  obtained  by  multiplying  by 
0-522. 

i6iA.    The  estimation  of  lactose  by  the  copper-iodide  method. 

The  method  is  exactly  similar  to  that  described  in  the 
previous  exercise.  The  author  is  responsible  for  the 


140 


THE    CARBOHYDRATES. 


[CH.    V. 


copper  valu  es  for  lactose .     They  are  represented  graphically 
in  fig.   1 8. 

It  must  be  noted  that  the  results  are  given  as  anhydrous 
lactose,  and  not  as  the  crystalline  hydrate. 


230160  90  ;•: 

:|;;i|i:|:;:|;|;;;:;;;;;;;;;;;:::; 

210  140  70  ::: 

'•"•'•  I:;::::  -  :  :  j  :  :  ::  : 

270200  130  60::: 

;;i:iij|::!jj|!i:|;:i:jii!:!:;;ji 

220150    80  10  -<  \\\\': 

210  140    70    pWrHrtl   Ilillll 
0               10 
100            110 
200           210 
30t)           310 

rM  JwKffit  f  rT4:|ffllllmj^ffil4^ 

20               30               40               50          '     60 
120            130            140            150            160 
220            230            240            250            26O 
320            330            340            350            860 

70               80               9O             10 

170            180            190           20 
270            280            290            3O 
370            380            390           40 

mg.  Cu. 
Fig.  18.    -Curve  showing  amount  of  copper  reduced  by  lactose  anhydride. 

In  the  case  of  lactose  as  much  as  250  mg.  may  be 
present  in  the  20  cc.  taken. 

The  copper  values  above  25  mg.  Cu.  can  be  converted 
to  anhydrous  lactose  by  the  use  of  the  following  formula  : 

mg.  anhydrous  lactose  =  1-25  +  mg.  Cu.  x  0-778. 


CH.   V.I  FEHLING'S    METHOD.  141 

62.    Fehling's  method. 

Preparation  of  solution.     See  Ex.  97,  p.  106. 

Method.  With  a  pipette  measure  10  cc.  of  freshly  prepared 
Fehling's  solution  into  a  small  flask.  Add  40  cc.  of  distilled  water, 
heat  the  mixture  till  it  boils  and  keep  it  boiling  the  whole  time.  Run 
in  the  sugar  solution  from  a  burette,  0-5  to  I  cc.  at  a  time,  allowing 
the  mixture  to  boil  for  about  15  sees,  between  each  addition.  A  red 
precipitate  of  cuprous  oxide  forms  and  the  intensity  of  the  blue  in 
the  supernatant  fluid  decreases.  Continue  to  add  the  sugar  till  this 
is  completely  removed.  This  is  best  determined  by  holding  the 
flask  by  the  rim  at  the  neck  and  viewing  it  by  transmitted  light.  If 
an  excess  of  sugar  be  added  a  yellow  or  brown  colour  appears  due  to 
the  formation  of  caramel  by  the  action  of  the  alkali  on  the  sugar. 

If  less  than  5  cc.  of  the  sugar  are  used,  the  solution  must  be 
diluted  till  about  10  cc.  are  necessary.  Thus  if  2-5  cc.  are  used 
in  the  first  rough  titration,  the  sugar  should  be  diluted  I  in  4,  by 
taking  25  cc.  and  adding  water  till  the  volume  of  the  solution  is 
100  cc.  The  burette  is  washed  out  and  filled  with  this  diluted 
solution  and  the  process  repeated.  But  this  time  run  in  nearly  the 
whole  of  the  sugar  solution  judged  necessary  at  such  a  rate  that  the 
mixture  does  not  go  off  the  boil.  Then  add  o-i  to  0-2  cc.  at  a  time 
till  the  reduction  is  complete.  This  titration  should  be  repeated  at 
least  once  more. 

Calculation.     10  cc.  of  Fehling's  solution  are  reduced  by  0-05  gram,  glucose. 

Example.     1-5  cc.  of  the  original  solution  necessary. 

Sugar  diluted  i  in  7  (10  cc.  sugar  made  up  to  70  cc.) 
10-2  diluted  sugar  solution  required  for  10  cc.  Fehling's. 
10-2  cc.  dil.  sugar  =  -05  gm.   glucose. 

•05  x  100     . 
ioo  cc.       „       „     =        I0.2 

TOO  cc.  original  sugar  =  '°5  x   IO°  x  7 

10-2 

=  3'43  per  cent. 

162 A.    Ling's  method. 

Preparation  of  the  indicator.  Dissolve  1-5  gram,  ammonium 
thiocyanate  and  i  gram,  ferrous  ammonium  sulphate  in  10  cc.  water 
at  about  45°  C.  and  cool  at  once.  Add  2*5  cc.  of  concentrated 


142  THE   CARBOHYDRATES.  [CH.   V. 

hydrochloric  acid.  The  solution  thus  obtained  has  invariably 
a  brownish-red  colour,  due  to  the  presence  of  some  ferric  salt.  Add 
zinc  dust,  in  small  portions  at  a  time,  till  the  fluid  is  just  colourless. 
On  standing  for  some  time  the  red  colour  reappears,  and  must  be 
removed  again  by  a  trace  of  zinc  dust.  But  the  delicacy  of  the 
indicator  is  impaired  by  being  decolourised  several  times.  When 
this  indicator  is  treated  with  a  cupric  salt,  the  colourless  ferrous 
thiocyanate  is  oxidised  to  the  red  ferric  thiocyanate. 

Method  of  analysis.  10  cc.  of  Fehling's  solution  and  about 
30  cc.  of  water  are  boiled  in  a  flask  and  the  sugar  solution  is  run  in 
from  a  burette  as  described  above  in  Fehling's  method.  The 
indicator  is  not  used  till  the  blue  colour  has  nearly  disappeared. 

Then  place  a  drop  of  the  indicator  on  a  white  slab.  Transfer 
a  drop  of  the  mixture  from  the  flask  to  the  middle  of  the  drop  of 
the  indicator  as  rapidly  as  possible  by  means  of  a  glass  tube.  If 
a  red  colour  appears  immediately  on  touching  the  drop  the  reduction 
is  not  completed.  More  sugar  must  be  added  and  a  fresh  drop  of  the 
indicator  used  as  before  till  no  colour  or  only  a  faint  tinge  of  red  is 
obtained.  If  less  than  5  cc.  of  the  sugar  solution  are  necessary  to  • 
complete  the  reaction,  the  solution  must  be  diluted  till  about  10  cc. 
are  required,  as  described  above  in  Fehling's  method. 

Special  precautions.  Use  a  glass  tube,  not  a  rod,  for  transferring 
the  drop. 

Do  not  put  your  finger  on  the  top  of  the  tube.  Dip  it  in  the 
flask  and  transfer  it  immediately  to  the  indicator.  The  flask  may 
be  taken  off  the  boil  for  an  instant  while  this  is  done. 

Do  not  stir  the  drops  on  the  slab. 

Wash  the  tube  before  using  it  again. 

Calculation  of  results.    This  is  the  same  as  in  Fehling's  method. 

163.    The  estimation  of  cane  sugar  by  Benedict's  method. 

Measure  50  cc.  of  the  solution  with  a  pipette  into  a  flask.  Add 
10  cc.  of  N.  hydrochloric  acid.  Boil  over  a  free  flame  and  keep 
the  mixture  very  gently  boiling  for  three  minutes.  Cool  under  the 
tap,  neutralise  by  the  addition  of  10  cc.  of  N.  sodium  hydroxide. 
Transfer  quantitatively  to  a  100  cc.  volumetric  flask  and  make  up 


CH.    V.]  POLARIMETER.  143 

the  volume  to  the  mark  with  cold  distilled  water,  rinsing  the  boiling 
flask  out  with  small  amounts  of  water.  Mix  carefully,  and  estimate 
the  invert  sugar  by  Benedict's  method. 

Calculation  of  results.  25  cc.  of  Benedict's  solution  =  0-0475 
gram,  hydrolysed  cane  sugar.  The  concentration  found  must  be 
multiplied  by  2,  owing  to  the  dilution  made  in  preparing  the 
hydrolysed  solution. 


E.    The  theory  and  use  of  the  Polarimeter. 

Waves  of  ordinary  light  vibrate  simultaneously  in  all 
directions  perpendicular  to  its  direction  of  propagation. 
By  means  of  certain  contrivances  it  is  possible  to  affect  the 
light  so  that  the  vibrations  proceed  in  a  single  plane.  Such 
light  is  plane  polarized.  The  plane  in  which  the  light  waves 
vibrate  is  called  the  plane  of  polarization.  This  conversion 
of  ordinary  light  into  polarized  light  is  generally  brought 
about  by  means  of  a  modified  prism  of  Iceland  spar  known 
as  a  Nicol's  prism.  If  a  beam  of  light  (fig.  19)  falls  on  the 


Fig.  19.      Crystal  of  Calc  Spar. 

face  of  a  rhombohedron  of  Iceland  spar  it  divides  on  enter- 
ing into  two  rays,  unequally  bent,  both  of  which  are 
polarized,  their  planes  of  polarization  being  at  right  angles 
to  one  another. 

The  extraordinary  ray  (E)  is  the  lesser  refracted  ray  : 
the  ordinary  ray  (O)  is  the  more  refracted  ray.  Before  the 
calc  spar  can  be  utilised  for  polariscopic  purposes  one  of  the 
rays  must  be  eliminated.  This  is  best  effected  by  Nicol's 
method  of  splitting  down  a  crystal  in  a  certain  plane,  grind- 
ing down  the  natural  ends  to  reduce  the  acute  angles  from 
71°  to  68°,  and  uniting  the  faces  by  Canada  balsam  (fig.  20). 


144 


THE    CARBOHYDRATES. 


[CH.    V 


A  beam  of  light  entering  parallel  to  the  long  sides  of  the 
prism  is  resolved  into  its  two  component  rays.  The  more 
refracted  (ordinary)  ray  (O)  meets  the  film  of  Canada 


c  D 

Fig.   20.       Diagram  of  refraction  in  a  Ni col's  prism. 

balsam  (CB),  and  is  completely  reflected  and  absorbed  by 
the  black  varnish  usually  placed  on  the  sides  of  the  prism. 
The  other  component  (the  extraordinary  ray)  (E)  passes 
through  the  film  of  balsam  and  emerges  in  a  polarized 
condition  from  the  end  surface  of  the'  Nicol. 


Fig.  21.     Plan  of  arrangement  of  a  simple  polarimeter. 

In  a  polarimeter  (fig.  2 1 )  a  second  Nicol  prism  called  an 
analyser  (A)  is  used  in  addition.  This  is  mounted  in  such  a 
way  that  it  can  be  rotated  around  its  long  axis.  The 
polarized  ray  that  emerges  from  the  polarising  Nicol  (P) 
falls  on  the  face  of  the  analyser,  and  will  only  pass  through 
unimpeded  provided  that  it  can  contrive  to  vibrate  in  the 
same  plane.  In  this  position  the  Nicols  are  said  to  be 
parallel.  If  the  analyser  be  rotated  through  an  angle  of 
45°  the  ray  is  completely  absorbed  and  the  Nicols  are  said 
to  be  crossed.  On  rotating  through  a  further  angle  Kof  45° 
the  Nicols  are  again  parallel.  Suppose  a  tube  of  water  be 
interposed  between  the  two  Nicols  (fig.  2 1 )  and  a  source  of 
light  at  L  be  viewed  through  the  system,  the  Nicols  are 
crossed  when  the  analyser  is  rotated  so  that  the  minimum 


CH.   V.] 


POLARIMETER. 


145 


illumination  is  obtained.  If  now,  instead  of  water,  the 
tube  be  filled  with  a  solution  of  glucose  or  of  certain  other 
substances,  it  will  be  found  that  the  illumination  is  not 
minimal.  To  attain  this  result  the  analyser  must  be 
rotated  through  a  certain  angle.  The  reason  for  this  is 
that  in  passing  through  the  glucose  solution  the  plane 

of  polarisation  has  been  gradually 
rotated  so  that  on  emerging  and 
striking  the  analyser  some  of  the 
light  can  get  through.  To  get  the 
minimum  illumination  the  analyser 
has  to  be  rotated  to  the  right  through 
an  angle  equal  to  that  through  which 
the  sugar  solution  has  rotated  the 
plane  of  polarisation  of  the  ray  that 
entered  it. 

The    simple     arrangement     de- 
scribed is  not  sufficiently  sensitive. 


O(D 


a 


Fig.  22.  Plan  of  a  three- 
field  polarimeter.  A  is 
the  polarising  Nicol  ; 
B  and  C  are  the  small 
accessory  Nicols  that 
resolve  the  field  into 
three  parts,  D,  E,  and 
F. 


Fig.  23.  The  appearances  seen  in  a  and  b  indi- 
cate that  the  analyser  is  not  in  the  correct 
position.  When  the  analyser  is  correctly 
adjusted  the  three  parts  of  the  field  have 
an  equal  feeble  illumination  as  shewn  in  c. 


Modern  instruments  have  two  small  Nicol's  prisms 
placed  between  the  polariser  and  the  solution  (see  fig. 
22),  the  effect  being  to  divide  the  field  into  three 
vertical  sections.  The  zero  and  end  points  are  obtained 
when  the  three  fields  have  an  equal  feeble  illumination 
(fig.  23,  c).  The  source  of  illumination  must  be  mono- 
chromatic, since  the  angle  of  rotation  varies  with  the  wave 
length  employed.  That  generally  used  is  sodium  light, 


146  THE   CARBOHYDRATES.  [CH.    V. 

obtained  by  heating  sodium  chloride  or  bromide  in  a  plati- 
num ring.  The  light  emitted  has  a  wave  length  correspond- 
ing to  the  D  line  of  the  solar  spectrum.  A  much  more 
brilliant  illumination  can  be  obtained  by  use  of  the  green 
rays  emitted  from  a  mercury  lamp.  The  rotation  being 
greater  with  the  shorter  wave  length,  greater  accuracy 
can  be  obtained.* 

The  rotation  varies  for  different  substances.  It 
is  increased  by  increasing  the  concentration  of  the 
solution  or  the  length  of  the  tube.  It  also  varies  with  the 
temperature,  nature  of  the  solvent,  and  the  wave-length  of 
the  light  used. 

The  specific  rotatory  power  is  the  rotation  observed 
through  a  tube  i  decimetre  in  length  of  a  solution  calculated 
to  be  100  per  cent.  This  is  generally  expressed  as  [a]. 
If  the  sodium  light  is  employed  it  is  expressed  as  [a]D. 

-        r  x  100 


where  r  =  the  observed  rotation. 

c  =  the  concentration  in  grams,  per  100  cc. 

/  =  the  length  of  the  tube  in  decimetres. 
If  the  temperature  is  defined,  it  is  usually  expressed  by 

[a]D20°. 

If   [a]D   be   known,    the   concentration   in    grammes   per 
100  cc.  is  given  by 

P  _  r  x  100 

WD  X  /' 

The  specific   rotatory  powers   of    the  more    common 
sugars  is  shewn  below.     I  am  indebted  to  Dr.   Lowry  for 


*  The  apparatus  in  the  author's  laboratory  consists  of  a  triple  field  instru- 
ment by  Hilger,  of  London,  fitted  with  a  horizontal  slit  and  a  direct  vision 
spectroscope.  A  mercury  lamp  is  used  as  the  source  of  illumination.  An 
accuracy  of  0-01°  is  easily  obtained. 


CH.    V.]  .  SPECIFIC    ROTATORY    POWER.  147 

the  information  concerning  the  rotations  of  the  sugars  to 
the  mercury  green.  In  all  cases  the  final  rotations  are 
given  (see  p.  103). 

WD  [fl]Hg 

Glucose..          ..  +  52'5  62 

Lactose  hydrate  ..      +  52*4  61*9 

Lactose  anhydride  +  55*2  65*2 

Maltose  . .          . .  +  138  163 

Sucrose  . .          . .  +  66*5  78*5 

Fructose             ..  ..  93-8             -  no'8 

d-Galactose        ..  +  81  95*7 

/-Xylose..          ..  +  19  22*4 

Invert  sugar     ..  ..  20-6  24*6 

For  carbohydrates  the  [a]Hg  can  be  obtained  by 
multiplying  [a]D  by  1*181.  This  relationship  is  not 
necessarily  true  for  substances  whose  molecular  construc- 
tion differs  from  that  of  the  carbohydrates. 

F.    Optical  Activity  and  the  Asymmetric  Carbon  Atom. 

If  C  be  a  carbon  atom  attached  to  a,  b  and  x,  different 
atoms  or  groups,  it  is  found  that  there  exists  only  one 
modification  of  the  type  Ca2bx.  But  if  the  structural 
formula  be  written  in  one  plane  it  would  appear  that  two 
arrangements  are  possible,  viz. 

a  a 


x  a 

A  B 

In  A  the  groups   a  are  adjacent,  whilst  in   B   they 
apparently  are  separated. 


148 


THE    CARBOHYDRATES. 


[CH.    V. 


The  accepted  explanation  of  the  facts  is  that  the  carbon 
atom  possesses  four  valencies  or  bonds  directed  towards 
the  apices  of  a  tetrahedron,  the  carbon  atom  being  at  the 
centre. 


The  student  is  advised  to  construct  such  a  model  from 
a  piece  of  plasticine  and  four  matches,  the  central  piece  of 
plasticine  representing  the  carbon  atom  and  the  matches 
the  four  bonds  attached  to  it.  The  heads  of  the  matches 
should  be  left  plain  or  marked  with  little  balls  of  variously 
coloured  plasticine  to  indicate  the  different  groups  attached 
to  the  central  carbon  atom.  Such  a  model  is  represented 
in  fig.  24. 

A 


Fig.  24.     Model  of  carbon  atom  attached  to  3  different  groups,  representing 
the  compound  Cazbx. 

Another  identical  model  should  now  be  prepared.  It 
will  be  found  that  the  two  can  be  superposed,  as  shewn 
in  fig.  25. 

Now  change  the  positions  of  any  two  matches  in  one 
of  the  models.  It  will  be  found  that  the  two  models  can 


CH.    V.]  ASYMMETRIC    CARBON    ATOMS.  149 

still  be  superposed.     In  fact,  no  matter  how  the  matches 

A 


Fig.  25.     Two  identical  superposable  models  of  the  type  Cazbx. 

are  changed  about  only  one  arrangement  is  possible.     This 


Fig.  26.     Model  of  an  asymmetric  carbon  atom  and  its  mirror  image. 

is  in  agreement  with  the  fact  that  only  one  modification 
of  the  type   Ca2bx  exists. 


150 


THE    CARBOHYDRATES. 


[CH.   V. 


Now  make  two  exactly  similar  models  in  each  of  which 
the  carbon  atom  is  represented  as  being  united  to  four 
different  groups.  Being  exactly  similar  the  two  models 
are  naturally  superposable.  Now  change  the  position  of 
any  two  matches  in  one  model  only.  The  two  models  thus 
formed  cannot  be  superposed.  On  examination  it  will 
be  found  that  the  two  models  have  a  relationship  to  one 
another  similar  to  that  of  the  right  to  the  left  hand,  or  of 
one  object  to  its  image  in  a  mirror.  This  is  represented 
in  fig.  26. 

If  now  still  another  model  be  constructed  it  will  be 
found  that  it  can  be  superposed  on  one  or  other  of  the  two 
previous  ones.  That  is,  there  exist  two  modifications 
and  two  only,  of  compounds  of  the  type  Cabxy. 

If  the  model  of  the  type  Ca2bx  be  examined  it  will  be 
seen  that  it  can  be  divided  into  two  symmetrical  halves. 
The  plane  of  symmetry  and  method  of  division  is  indicated 
in  fig.  27.  It  must  be  understood  that  though  a  plane  of 
symmetry  exists  the  atoms  or  groups  are  not  actually  split 
into  halves  by  it.  An  examination  of  the  figures  shewn 

in  fig.  26  will  reveal  the 
fact  that  they  do  not 
possess  a  plane  of  sym- 
metry. It  has  been  ascer- 
tained that  all  compounds 
of  the  type  Cabxy  exist 
in  two  modifications.  The 
solutions  of  one  of  these 
rotates  the  plane  of  polari- 
sation to  the  right  :  that 
of  the  other  exactly  the 
same  degree  to  the  left. 
The  former  is  the  "dextro- 
rotatory" or  the  ^-com- 
pound :  the  latter  is  the 
"laevo-rotatory"  or  /-com- 
pound. These  are  sometimes  known  as  enantiomorphs. 
Since  the  compound  has  no  plane  of  symmetry  a  carbon 


Fig.  27. 


Plane  of  symmetry  of  model 
shewn  in  Fig.  24. 


CH.   V.] 


ASYMMETRIC   COMPOUNDS. 


151 


atom  attached  to  four  different  groups  is  known  as  an 
asymmetric  carbon  atom.  The  possession  of  an  asymmetric 
carbon  atom  in  a  compound  is  essential  to  the  possession 
of  optical  activity  by  that  compound. 

If  equal  parts  of  the  d-  and  /-  varieties  of  a  compound 
be  mixed  together,  the  solution  of  the  substance  is  "opti- 
cally inactive  by  external  compensation."  Such  an  in- 
active mixture  is  known  as  "racemic,"  and  is  designated 
by  dl-  or  i-.  When  a  compound  that  contains  an  asym- 
metric carbon  atom  is  synthesised,  it  is  always  found  that 
equal  parts  of  the  d-  and  /-  varieties  are  formed.  These 
can  often  be  resolved  into  their  active  constituents  by 
various  methods,  the  most  interesting  of  which  is  the 
biochemical,  depending  on  the  property  of  living  organisms 
of  selective  assimilation,  one  of  the  two  components  being 
destroyed  more  rapidly  than  the  other.  A  few  examples 
of  this  are  given  below. 


Substance 

Organism 

Destroyed 

Lactic  acid 

penicillium 

d. 

bacteria 

I. 

Glyceric  acid 

penicillium 

I. 

bac.  ethaceticus 

I. 

Amyl  alcohol 

fungus 

I. 

Glucose,  mannose, 

galactose,  fructose 

yeast 

d. 

Racemic  acid 

penicillium 

d. 

*  i     * 

schizomycetes 

I. 

Leucine 

yeast 

I. 

Alanine 

yeast 

d. 

This  power  of  selective  assimilation  finds  a  parallel  in 
the  different  physiological  action  of  enantiomorphs  on  the 
body,  and  of  the  body  and  also  of  enzymes  on  enantio- 
morphs. For  instance,  ^/-adrenaline  has  only  slightly 
more  than  one-half  the  physiological  activity  of  the  natural 


152  THE    CARBOHYDRATES.  [cH.    V. 

/-adrenaline :  ^-asparagine  has  a  sweet  taste,  whilst  /-aspara- 
gine  is  insipid  :  /-nicotine  is  far  more  poisonous  than 
^-nicotine.  Further,  if  d/-phenyl-aminoacetic  acid  be 
administered  to  an  animal,  only  the  /-  variety  is  found  in 
the  urine,  the  body  having  the  power  to  destroy  the 
d-acid.  Many  other  similar  instances  have  been  described. 

Proteins,  like  casein,  can  be  racemized  by  treatment 
with  dilute  alkalies.  Such  proteins  are  not  attacked  by 
the  pro teoly tic  enzymes,  and  when  administered  to  dogs 
can  be  recovered  quantitatively  from  the  faeces.  The 
relationship  between  configuration  and  enzyme  action 
is  discussed  on  p.  1 84. 

In  a  few  cases  substances  having  two,  four  or  six 
asymmetric  carbon  atoms  are  optically  inactive,  and  can- 
not be  resolved  into  two  components.  The  optical 
inactivity  is  due  to  internal  compensation,  the  molecule 
possessing  a  plane  of  symmetry.  The  simplest  example 
of  this  is  that  of  mesotartaric  acid.  We  can  represent  the 
formulae  of  the  tartaric  acids  in  one  plane  as  follows  :— 

COOH  COOH  COOH 

I  !  I 

HO— C— H  H— C— OH  H— C— OH 


HO— C— H  H— C— OH 

I  I  I 

COOH  COOH  COOH 

A.  B.  C. 

d-Tartaric  Acid.      /-Tartaric  Acid.       Mesotartaric  Acid. 

The  dotted  line  in  the  structural  formula  of  Mesotartaric 
Acid  indicates  the  plane  of  symmetry.  A  can  be  regarded 
as  the  mirror  image  of  B.  Admixture  of  these  in  equal 
parts  will  be  inactive  through  external  compensation.  If 
the  upper  carbon  atom  of  C  be  regarded  as  dextro-rotatory, 
then  the  lower  one  can  be  regarded  as  its  mirror  image 
and  will  therefore  be  laevorotatory.  The  whole  molecule 
will  therefore  be  optically  inactive,  and  the  compound  is 
incapable  of  being  resolved  into  two  constituents. 


CHAPTER  VI. 
THE   FATS,   OILS  AND   LIPINES. 

These  compounds  are  characterised  physically  by 
having  low  melting  points,  a  greasy  feel,  and  by  being 
insoluble  in  water ;  but  soluble  in  ether,  alcohol,  chloroform, 
and  certain  other  organic  solvents,  and  chemically  by  being 
mainly  composed  of  the  radicles  of  the  higher  fatty  acids. 
A  certain  number  of  substances  with  the  appropriate 
physical  properties  have  been  found  to  belong  chemically 
to  the  terpenes. 

The  following  classification  (after  Gies)  include  all 
those  of  known  physiological  interest : 

1.  Fats.     Neutral  glycerides  of  fatty  acids,  solid  at 

20°C. 

2.  Fatty  oils.     Neutral  glycerides  of  fatty  acids,  liquid 

at  2o°C. 

3.  Essential  oils.     Volatile  substances  of  varied  chemi- 

cal nature,  e.g.  oil  of  cloves. 

4.  Sterols.     Alcohols  of  the  terpene  group,  e.g.  choles- 

terol. 

5.  Waxes.     Esters  of  sterols  and  fatty  acids,  e.g.  bees- 

wax and  spermaceti. 

6.  Phospholipins    or     phosphatides.        Compounds    of 

fatty  acids  containing   phosphorus  and  nitro- 
gen, e.g.  lecithin,  kephalin. 

7.  Galactolipins  or  cerebrosides.       Compounds  of  fatty 

acids,  galactose  and  a  nitrogenous  complex. 


The  fats  and  fatty  oils  are  glycerol  esters  of  the  higher 
fatty  acids. 


154  FATS,    OILS   AND    LIPINES.  [cH.  VI. 

An  ester  is  a  compound  formed  by  the  condensation  of 
an  alcohol  with  an  acid.  Glycerol,  being  a  trivalent 
alcohol,  can  condense  with  three  molecules  of  a  fatty  acid. 

CH2OH  HOOC.  X  CH2.OOC.  X 

CH.OH     +      HOOC.  Y    =      CH.OOC.  Y    +  3H2O 

CH2.OH  HOOC.  Z  CH2.OOC.  Z 

Glycerol.  Fatty  acids.         Neutral  fat  or 

fatty  oil. 

The  three  radicles,  X,  Y  and  Z  may  be  the  same,  or  they 
may  differ. 

The  fatty  acids  most  commonly  found  in  the  composi- 
tion of  these  substances  are 

Palmitic  acid,  C15H31.COOH 

or  CH3.(CH2)14.COOH. 

Stearic  acid,      C17H35.COOH 

or  CH3(CH2)16.COOH. 

Oleic  acid,         C17H33.COOH 

or  CH3(CH2)7.  CH  =  CH.(CH2)7.COOH. 

As  can  be  seen  from  the  formulae,  the  first  two  are 
saturated  acids  of  the  acetic  acid  series,  whilst  oleic  acid 
is  unsaturated  and  belongs  to  the  acrylic  acid  series. 
Occasionally  other  acids,  more  unsaturated  than  oleic  acid, 
are  found,  such  as  linoleic  acid. 

Palmitic  acid  melts  at  62-6°C.  ;  stearic  acid  at  69'3°C ; 
oleic  at  i4°C.,  solidifying  at  4°C.  They  are  all  insoluble  in 
water,  and  only  slightly  soluble  in  cold  alcohol,  with  the 
exception  of  oleic  acid,  which  dissolves  readily.  They  are 
all  freely  soluble  in  ether.  The  sodium  and  potassium 
salts  of  these  acids  are  known  as  soaps,  which  are  readily 
soluble  in  water.  If  these  salts  be  diluted  with  water  they 
are  hydrolytically  dissociated. 

NaA  +  HOH  =  NaOH  +    HA 
Soap.  Fatty  acid. 


CH.   VI.]  PROPERTIES   OF  FATS.  155 

Since  the  fatty  acid  is  only  slightly  ionised,  and  is  in- 
soluble in  water,  dilution  of  a  clear  solution  of  sodium 
stearate  causes  the  liberation  of  sodium  hydroxide  and  of 
the  fatty  acid,  the  latter  causing  the  solution  to  become 
opalescent.  This  reaction  is  not  so  well  marked  with  the 
oleates.  This  hydro ly tic  dissociation  is  checked  by  the 
addition  of  alcohol.  For  this  reason  it  is  essential  to  have 
alcohol  present  to  the  extent  of  50  per  cent,  at  the  end  of  a 
titration  of  fatty  acids  with  aqueous  alkalies. 

The  calcium,  magnesium,  barium  and  lead  salts  or 
soaps  are  insoluble  in  water. 

The  glycerides  formed  by  these  three  acids  are  known  as 
tripalmitin,  tristearin  and  triolein  respectively.  The 
melting  points  are  66°,  71°  and  —  5°C.  In  the  body  they 
are  found  mixed  in  different  proportions,  and  possibly 
some  of  the  compounds  have  more  than  one  fatty  acid  in 
the  molecule.  They  are  hydro lysed  by  boiling  acids  and 
alkalies,  by  superheated  steam,  and  by  certain  enzymes 
called  lipases  or  steapsins.  If  an  alkali  be  used  as  the 
hydrolytic  reagent,  the  fatty  acid  liberated  combines  with 
the  alkali  to  form  a  soap.  This  special  form  of  hydrolysis 
is  therefore  called  saponification.  The  most  rapid  method 
of  effecting  this  hydrolysis  in  the  laboratory  is  by  boiling 
the  fat  with  an  alcoholic  solution  of  soda  or  potash.  The 
fat  being  soluble  in  the  alcohol  the  formation  of  ethyl  esters 
of  the  fatty  acids  proceeds  rapidly.  The  ethyl  esters  are 
themselves  hydrolysed  by  the  water  present,  and  so  the  fat 
is  completely  converted  into  glycerol  and  soap. 

Various  methods  have  been  devised  for  the  identifica- 
tion of  the  fats  and  oils,  amongst  them  being. 

1.  The  melting  point,  or  the  solidifying  point. 

2.  The  saponification  value.     A  known  weight  of  the 
fat  is  heated  with  a  given  volume  of  a  standardised  alco- 
holic solution  of  potash.     The  mixture  is  then  titrated  with 
standard    hydrochloric    acid    against    phenol    phthalein, 
alcohol  being  added  to  maintain  a  concentration  of  50  per 
cent.     The  number  of  milligrammes  of  potassium  hydroxide 


156  FATS,   OILS  AND  LIPINES.  [CH.    VI. 

that  have  been  neutralised  by  the  free  or  combined  fatty 
acids  of  i  gram,  of  the  fat  is  the  value  required. 

The  saponification  value  of  pure  tristearin  is  189. 
(C17H35.COO)3C3H5  +  3KOH 

=  3C17H35.COOK  +  C3H5(OH)3 
Mol.  wt.  890  3  x  56 

i   gram,  requires  0-189  gram.   KOH. 

The  value  for  triolein  is  190,  and  for  tripalmitin  208. 

The  saponification  value  is  a  measure  of  the  mean 
molecular  weight  of  the  fatty  acids  constituting  the  fat. 
It  is  increased  by  a  decrease  in  the  molecular  weight.  It  is 
lowered  by  the  presence  of  unsaponifiable  substances,  such 
as  cholesterol. 

3.  The  iodine  value.  Oleic  acid  is  an  unsaturated 
acid,  and  can  combine  with  two  atoms  of  iodine.  The 
saturated  acids  and  their  glycerides  do  not  absorb  iodine. 
The  iodine  value  is  the  grams,  of  iodine  absorbed  by  100 
grams,  of  the  fatty  material.  Thus,  triolein  has  a  mole- 
cular weight  of  884,  and  can  absorb  6  atoms  of  iodine  per 
molecule.  So  that  884  grams,  absorb  6  x  127  =  762  grams, 
of  iodine,  or  86-2  per  cent.  Since  some  fats  contain 
radicles  with  more  than  one  double  bond,  it  is  clear  that  the 
iodine  value  will  not  determine  absolutely  the  character  of  a 
fat. 

The  emulsification  of  the  fats. 

Fats  can  be  emulsified,  i.e.  broken  up  into  droplets, 
either  mechanically  by  agitation,  or  "  spontaneously." 

"  Spontaneous  "  emulsification  takes  place  when  a 
melted  oil  or  fat  that  contains  a  certain  percentage  of  free 
fatty  acid  is  brought  into  contact  with  an  alkali.  The 
fatty  acid  dissolves  in  the  alkali  to  form  a  soluble  soap,  and 
the  diffusion  currents  thus  set  up  break  the  globule  of  fat 
into  small  particles,  the  process  being  maintained  by  the 
continual  exposure  of  fatty  acid  to  the  alkali.  The 


CH.   VI.]  EMULSIFICATION.  157 

fat  in  the  small  intestine  is  thus  emulsified  as  a  preliminary 
to  complete  hydrolysis  by  the  pancreatic  lipase. 

The  digestion  of  fats. 

The  fats  are  hydrolysed  to  a  small  extent  in  the  stomach 
by  gastric  lipase.  This  action  is  greater  if  the  fat  be  given 
in  an  emulsified  form,  as  in  milk. 

In  the  duodenum,  the  fat  mixed  with  the  fatty  acid  is 
spontaneously  emulsified  by  the  alkaline  bile,  succus 
entericus  and  pancreatic  juice.  The  emulsified  fat  is  then 
completely  hydrolysed  to  glycerol  and  fatty  acids  by  the 
pancreatic  lipase.  The  fatty  acids  are  converted  into 
soluble  soaps  by  the  alkalies  present.  The  soaps  and 
glycerol  are  absorbed  into  the  epithelial  cells  bordering  the 
villi,  where  they  are  resynthesised  into  fats.  These  are 
passed  into  the  lacteals,  and  reach  the  blood  stream  by  way 
of  the  thoracic  duct. 

164.  (a)  Carefully  allow  a  drop  of  neutral  olive  oil  to  fall 
gently  on  to  the  surface  of  some  0*25  per  cent,  sodium  carbonate 
contained  in  a  watch-glass.  The  drop  of  oil  remains  quite  clear,  and 
forms  a  thin  (circular)  film  on  the  surface. 

(b)  Shake  4  cc.  of  neutral  oil  with  3  drops  (only)  of  oleic  acid  in  a 
dry  test-tube.     With  a  drop  of  this  mixture  repeat  (a),  using  a 
fresh  watch-glass  full  of  Na2CO3.    The  rancid  oil  slowly  spreads  out 
in  an  amoeboid  fashion  and  becomes  converted  into  a  milky  emul- 
sion. 

(c)  To  the  remainder  of  the  mixture  of  oil  and  oleic  acid  add 
12  more  drops  of  oleic  acid,  shake  well,  and  repeat  the  experiment. 
The  drop  becomes  white  and  opaque,  but  does  not  become  emulsified. 

NOTES. — i .  It  is  absolutely  essential  that  the  oil  be  quite  neutral,  and  this 
can  best  be  tested  by  dropping  it  on  to  0-25  per  cent.  Na2CO3.  If  a  spontaneous 
emulsion  is  formed,  a  fresh  sample  must  be  obtained,  or  melted  fresh  butter 
substituted. 

2.  The  spontaneous  emulsion  in  (b)  is  caused  by  the  trace  of  oleic  acid 
dissolving  in  the  alkali  to  form  a  soap,  diffusion  currents  being  thus  set  up 
which  divide  the  fat  into  microscopic  droplets. 

3.  It  is  important  to  mix  the  oil  very  thoroughly  with  the  oleic  acid. 

4.  In  (c)  the  large  excess  of  oleic  acid  leads  to  the  opaque  ring  of  soap 
being  formed  round  the  oil,  and  this  soap,  being  only  slightly  soluble  in  water, 
prevents  the  formation  of  an  emulsion. 


158  FATS,   OILS  AND    LIPINES.  [cH.   VI. 

165.  Shake  a  few  drops  of  olive  oil  with  5  cc.  of  ether  in  a 
dry  tube.    The  oil  completely  dissolves.     Repeat  the  experiment 
with  alcohol  instead  of  ether.    The  oil  dissolves  partially,  but  is  not 
so  soluble  in  alcohol  as  in  ether.     Pour  the  alcoholic  solution  into 
water.     The  fat  is  precipitated  as  an  emulsion. 

166.  Touch  a  piece  of  writing  paper  with  a  glass  rod  that  has 
been  dipped  in  olive  oil.    The  paper  is  rendered  translucent. 

Preparation  of  pancreatic  lipase.  A  perfectly  fresh  pig's  pancreas  is 
freed  from  fat,  weighed,  finely  minced  and  ground  with  sand.  It  is  then 
treated  with  three  times  its  weight  of  water  and  its  own  weight  of  strong  alcohol. 
It  is  allowed  to  stand  for  three  days  at  room  temperature  and  strained  through 
muslin.  It  must  not  be  filtered.  When  not  in  use  it  should  be  kept  in  a 
refrigerator.  It  will  remain  active  for  a  considerable  time. 

NOTE. — Pancreatic  lipase  is  a  ferment  that  only  acts  with  the  co-operation 
of  a  co-ferment,  which  is  soluble  in  water  and  not  destroyed  by  boiling.  Bile 
salts  and  certain  other  substances  can  act  as  the  co-ferment.  The  ferment 
proper  is  practically  insoluble  in  water,  and  is  destroyed  by  boiling.  If  the 
pancreatic  extract  be  filtered,  neither  the  precipitate  nor  the  filtrate  has  any 
appreciable  action  on  fats  ;  but  when  the  two  are  mixed  the  original  lipolytic 
action  is  recovered.  The  precipitate  is  the  ferment ;  the  filtrate  contains  the 
co-ferment. 

Preparation  of  an  Emulsion  of  Fat.  —  Commercial  olive  oil  (which 
contains  some  free  oleic  acid)  is  treated  in  a  flask  with  i  drop  of  a  i  per  cent, 
alcoholic  solution  of  phenolphthalein  for  every  10  cc.  of  oil.  Decinormal 
sodium  hydroxide  is  added,  with  frequent  shaking,  till  the  mixture  is  slightly 
alkaline,  as  shewn  by  a  very  faint  pink  tinge.  A  very  stable  emulsion  is  thus 
formed,  and  thus  a  considerable  surface  of  fat  is  exposed  to  the  action  of  the 
ferment. 

Fat-splitting  action  of  lipase  (steapsin). 

167.  Label  three  test-tubes  A,  B,  and  C. 

To  A  add  2  cc.  of  the  pancreatic  extract  and  i  cc.  of  water. 
,,    B  ,,  „  „  ,  boil  and  add  i  cc.  of 

water. 
„    C  „  „  „  and  i  cc.  of  i  per  cent. 

bile  salts. 

It  is  advantageous  to  have  the  tubes  fitted  with  well-fitting  rubber 
stoppers.  To  each  add  5  cc.  of  the  emulsion  of  oil,  shake  thoroughly, 
and  place  in  a  water  bath  at  40°  C.  for  i  hour.  Shake  the  tubes 
thoroughly  every  fifteen  minutes.  At  the  end  of  the  digestion 
transfer  the  contents  to  three  labelled  beakers.  Add  10  cc.  of  96 
per  cent,  alcohol  to  the  tube,  shake  well,  and  transfer  the  alcoholic 


CH.    VI.]  LIPASE.  159 

washings  to  its  appropriate  beaker.  Repeat  this  with  another  10  cc. 
of  alcohol.  To  each  beaker  add  5  drops  of  i  per  cent,  phenol 
phthalein  and  titrate  with  o-i  N.  NaOH  to  a  faint  definite  pink. 
The  results  vary  considerably  with  different  preparations,  but  the 
following  may  be  taken  as  an  example : 

A  required    6-7  cc.  of  o-i  N.  NaOH 
B         „          2-3 
C         „        14-9 

6-7  —  2-3  =    4-4  is  a  measure  of  the  amount  of  fatty  acid  produced 

in  A. 

14-9  —  2 -3  =  12-6  is  a  measure  of  the  amount  of  fatty  acid  produced 

in  C. 

It  will  be  found  that  the  presence  of  the  bile  salts  materially 
aids  the  digestion  of  the  fat. 

1 68.  Detection  of  lipase.  Boil  about  5  cc.  of  milk  to  destroy 
bacilli  that  may  ferment  the  lactose.  Cool  and  add  2  cc.  of  the 
pancreatic  extract.  Add  about  i  cc.  of  a  o-oi  per  cent,  solution  of 
phenol  red  (see  p.  24),  and  then  enough  of  a  2  per  cent,  solution  of 
sodium  carbonate  to  give  the  solution  a  distinct  reddish  tinge. 
Divide  into  two  portions,  A  and  B.  Boil  B,  and  then  cool  it  under 
the  tap.  Place  the  tubes  in  a  warm  bath  at  40°  C.,  and  examine 
at  intervals.  A  will  become  yellow  if  lipase  is  present,  owing  to  the 
hydrolysis  of  the  emulsified  fat  of  the  milk  into  fatty  acids. 

NOTE. — The  hydrolysis  of  casein  by  trypsin  leads  to  a  slight  increase  in 
the  concentration  of  hydrogen  ions.  For  that  reason  it  is  preferable  to  use  a 
mixture  of  cream  and  water  instead  of  milk. 


Glycerol. 

169.  Treat  a  drop  or  two  of  glycerol  in  a  test-tube  with  a 
solution  of  copper  sulphate  and  then  with  sodium  hydroxide.    A 
blue  solution  is  obtained,  glycerol  preventing  the  precipitation  of 
cupric  hydroxide.     (See  Ex.  96,  note  3.) 

170.  Boil  the  solution  thus  obtained.     Reduction  does  not 
occur. 


160  FATS,    OILS   AND   LIPINES.  [cH.    VI. 

171.  Heat  strongly  a  drop  or  two  of  pure  glycerol  with  solid 
potassium  hydrogen  sulphate  in  a  dry  test-tube.    The  pungent 
odour  of  acrolein  (acrylic  aldehyde)  is  noticed. 

CH2OH.CHOH.CH2OH  =  CH2 :  CH.CHO  +  2H2O 

Glycerol.  Acrolein. 

172.  Treat  about  5  cc.  of  a  0-5  per  cent,  solution  of  borax 
with  sufficient  of  a  I  per  cent,  alcoholic  solution  of  phenolphthalein 
to  produce  a  well-marked  red  colour.     Add  a  20  per  cent,  aqueous 
solution  of  glycerol  drop  by  drop,  until  the  red  colour  is  just  dis- 
charged.    Boil  the  solution :  the  colour  returns,  provided  that  an 
excess  of  glycerine  has  not  been  added  (Dunstan's  test  for  glycerol). 

NOTES. — i.     Any  ammonium  salt  will  discharge  the  colour,  but  in  this 
case  it  does  not  return  on  heating. 

2.  Any  polyhydric  alcohol  is  likely  to  give  the  same  reaction.     The  sugars 
are  all  polyhydric  alcohols,  but  are  distinguished  from  glycerol  by  their  reducing 
properties,  etc.,  and  by  the  fact  that  they  are  not  volatile  when  distilled  by 
steam. 

3.  The  probable  explanation  of  the  reaction  is  as  follows.     Sodium  borate 
is  partially  hydrolysed  in  aqueous  solution  to  boric  acid  and  sodium  hydroxide. 
Boric  acid  being  a  weak  acid  is  only  feebly  ionised  and  therefore  the  solution 
reacts  alkaline.     On  adding  glycerol,  glyceroboric  acid  is  formed.     This  is  a 
strong  acid  and  hence  the  reaction  of  the  solution  changes  from  alkaline  to  acid. 
On  heating,  unless  a  large  excess  of  glycerol  be  present,  the  glyceroboric  acid 
is  hydrolysed  to  glycerol,  and  boric  acid,  and  the   solution  again  becomes 
alkaline. 


The  Higher  fatty  acids  and  their  salts,  the  soaps. 

173.  Shake  a  few  drops  of  oleic  acid  with  5  cc.  of  water,  ether 
and  alcohol  respectively  in  separate  tubes.    The  acid  is  insoluble  in 
water,  but  soluble  in  alcohol  or  ether. 

174.  Place  a  drop  of  oleic  acid  on  writing  paper :  a  greasy  stain 
results. 

175.  Shake  the  alcoholic  solution  of  oleic  acid  with  dilute 
bromine  water.     The  colour  of  the  bromine  is  discharged,  owing  to 
the  unsaturated  acid  absorbing  the  halogen  till  it  is  saturated. 

176.  Repeat  the  experiment  with  an  alcoholic  solution  of 
stearic  acid  or  commercial  "  stearine  "  (a  mixture  of  stearic  and 
palmitic  acids).    The  colour  of  the  bromine  persists,  since  these  acids 
are  members  of  the  saturated  series. 


CH.    VI.]  SOAPS.  161 

177.  To  about  10  drops  of  oleic  acid  add  10  cc.  of  boiling  dis- 
tilled water,  and  to  the  hot  mixture  add  10  per  cent.  NaOH  drop  by 
drop  till  the  solution  is  clear.     If  an  excess  be  added  the  excess  of 
sodium  ions  causes  a  precipitate  (see  note  below) .     A  clear  solution  of 
a  soap,  sodium  oleate,  is  formed.     Divide  this  into  three  portions. 
To  A  add  a  few  drops  of  strong  HC1  or  H2SO4  till  the  reaction  is 
distinctly  acid.     Oleic  acid  separates  out  and  rises  to  the  surface 
of  the  tube. 

To  B  add  finely-powdered  sodium  chloride  and  shake.  The 
soap  is  rendered  insoluble  and  rises  to  the  surface. 

To  C  add  some  calcium  chloride.  A  precipitate  of  an  insoluble 
soap,  calcium  oleate,  is  produced. 

NOTE. — B  illustrates  the  principle  of  "  salting  out,"  which  is  used  in  the 
manufacture  of  soaps.  The  excess  of  sodium  ions  in  the  solution,  produced 
by  the  addition  of  the  sodium  chloride,  lowers  the  solubility  of  the  sodium 
oleate,  which  is  therefore  precipitated. 

178.  Boil  2  cc.  of  olive  oil  with  5  cc.  of  a  20  per  cent,  alcoholic 
solution  of  sodium  hydroxide  in  a  basin  over  a  small  flame  for  five 
minutes  or  until  the  alcohol  has  all  evaporated  away.    Add  about 
5  cc.  of  alcohol  and  heat  again  to  dryness,  stirring  the  whole  time. 
Add  about  50  cc.  of  distilled  water  and  boil  till  dissolved.     Add 
solid  sodium  chloride  and  stir.     The  soap  formed  is  precipitated. 
Filter  some  off,  dissolve  in  boiling  water  and  repeat  the  experiments 
A,  B,  and  C,  described  in  the  previous  exercise. 

Cholesterol.  C^H^OH  or  C27H45OH  is  a  secondary 
alcohol,  which  is  very  widely  distributed  in  animal  tissues. 
An  isomer,  known  as  phytosterol,  is  found  in  many  veget- 
able oils.  It  was  first  discovered  in  gall  stones,  hence  its 
name.  A  considerable  amount  is  found  in  nervous  tissues 
and  in  egg  yolk.  In  blood  serum  it  is  present  as  an  ester, 
as  it  is  in  "  lanoline,"  the  fatty  matter  obtained  from 
sheep's  wool.  It  is  readily  soluble  in  acetone,  chloroform, 
ether  and  benzene.  It  is  only  slightly  soluble  in  cold,  but 
easily  soluble  in  hot  alcohol.  It  is  soluble  in  the  bile  salts. 
It  is  insoluble  in  water,  weak  acids,  and  alkalies. 

It  crystallises  from  hot  alcohol  in  rhombic  plates, 
which  often  have  a  re-entering  (notched)  angle.  From  dry 
ether,  chloroform  and  benzene  it  crystallises  in  needles. 


162  FATS,    OILS   AND   LIPINES.  [cH.    VI. 

It  melts  at  i45°C.     In  chloroform  solution  it  is  laevorota- 
tory,  [a]  =  -36-6°. 

Its  chemical  constitution  is  not  yet  determined,  but 
it  is  probably  a  member  of  the  terpene  series. 

179.  Preparation  of  cholesterol  from  sheep's  brain.    Sheep's 
brain  is  minced,  ground  with  sand,  and  intimately  mixed  with  three 
times  its  weight  of  plaster  of  paris.     After  some  hours  the  hard  mass 
is  ground  and  extracted  three  times  with  cold  acetone  by  rubbing 
well  in  a  mortar.    The  mixed  acetone  solutions  are  filtered  and 
allowed  to  evaporate  spontaneously.    The  cholesterol  crystallises 
out  and  is  recrystallised  from  boiling  alcohol. 

1 80.  Mount  a  few  crystals  of  cholesterol  in  water,  examine 
under  the  microscope,  and  draw  them.     Note  the  rhombic  plates, 
which  are  often  incomplete  at  one  corner.     Irrigate  the  crystals 
with  strong  sulphuric  acid  :  they  turn  red  at  the  edges.     Now  add  a 
drop  of  iodine  solution  :  the  crystals  give  a  violet  colour,  changing 
lo  a  green,  blue,  and  finally  a  black. 

181.  Salkowski's  reaction  for  cholesterol.     Dissolve  a  little 
in  a  few  cc.  of  chloroform ;   to  the  soution  add  an  equal  quantity 
of  strong  sulphuric  acid  and  shake.     The  upper  layer  of  chloroform 
becomes  red,  the  layer  of  sulphuric  acid  yellow  with  a  green  fluor- 
escence. 

182.  Liebermann-Burchard  reaction  for   cholesterol.       Dis- 
solve a  little  cholesterol  in  2  cc.  of  chloroform,  contained  in  a 
perfectly  dry  tube.     Add  ten  drops  of  acetic  anyhdride,  then  two 
drops  of  strong  sulphuric  acid,  and  shake.    The  solution  becomes 
coloured  a  deep  blue. 

Phospholipins  or  Phosphatides.  As  mentioned  on  p.  153, 
these  are  compounds  of  fatty  acids  with  phosphorus  and 
nitrogen.  Maclean*  classifies  them  as  follows  :— 

(A)     Monaminophosphatides  (N  :  P  =  i  :  i) 

(a)  Lecithin. 

(b)  Kephalin. 

*  Lecithin  and  Allied  Substances,  by  H.  Maclean  (Longmans,  Green  & 
Co.,  1918). 


CH.    VI.]  LECITHIN.  163 

(B)  Diaminophosphatides  (N  :  P  =  2  :  i) 

Sphingomy  elin . 

(C)  Monaminodiphosphatides  (N  :  P  =  i  :  2) 

Cuorin. 

Their  separation  from  other  constituents  of  tissues  is 
dependent  on  the  fact  that  though  they  are  soluble  in  ether 
they  are  insoluble  in  acetone. 

Lecithin  is  the  best  known  member  of  the  series. 
The  following  account  of  its  properties  is  abridged  from 
Maclean's  valuable  monograph.  It  is  a  yellowish-white 
waxy  substance,  which  on  exposure  to  the  air  absorbs 
oxygen  and  soon  assumes  a  dark  brown  colour.  It  is  very 
hygroscopic  and  in  the  presence  of  moisture  forms  a  soft 
plastic  mass.  It  dissolves  very  easily  in  alcohol,  ether, 
chloroform,  benzene,  petroleum  ether  and  many  other 
organic  reagents  :  also  in  aqueous  solutions  of  bile  salts. 
It  is  insoluble  in  acetone  and  methyl  acetate.  In  contact 
with  water  it  swells  up  and  ultimately  forms  a  slimy 
emulsion  or  colloidal  solution,  from  which  it  is  readily 
precipitated  by  salts  with  divalent  cations,  such  as  calcium 
and  magnesium ;  salts  containing  monovalent  cations, 
such  as  sodium  chloride,  act  in  the  same  way,  but  more 
slowly.  In  the  presence  of  a  small  amount  of  sodium 
chloride,  acetone  readily  precipitates  lecithin  from  its 
emulsions  with  water.  It  is  also  readily  precipitated  from 
ether  or  chloroform  solution  by  this  reagent.  On  treatment 
with  alkalies  or  acids  it  is  hydrolysed,  quickly  on  heating 
and  more  slowly  in  the  cold.  Lecithin  combines  with 
acids  and  bases.  It  also  combines  with  certain  salts  of  the 
heavy  metals,  such  as  cadmium  chloride,  platinum  chloride, 
and  mercuric  chloride.  Lecithin-cadmium-chloride  is 
almost  insoluble  in  alcohol,  but  dissolves  in  a  mixture  of 
carbon  disulphide  and  ether  or  alcohol.  It  is  probable 
that  the  greater  part  of  the  lecithin  of  tissues  exists  in  some 
kind  of  combination  with  protein.  Leithin  is  dextro- 
rotatory. 


164 


FATS,    OILS   AND   LIPINES. 


[CH.   VI. 


The   constitution   of    lecithin   can   be   represented   as 
follows  : — 

-" Tatty  acid  radicle. 

Glycerol Fatty  acid  radicle. 

acid  radicle — choline. 


It  can  be  regarded  as  a  complex  of  glycero-phosphoric 
acid  with  fatty  acids  and  with  choline. 

Glycero-phosphoric  acid  is 
CH2.OH 


H.OH 
CH2— O 
HO— P=O 
OH 


Choline  is 


N- 


C2H4.OH 

:(CH3)3 
(OH) 


so  that  if  the  fatty  acids  be  represented  by  R.COOH,  the 
constitutional  formula  of  lecithin  may  be 

CH2.OOC.R. 
CH.  OOC.R. 
CH2— O 

i 

HO— P=O 


N 


E(CH3)3 
OH 


CH.    VI.]  CEREBROSIDES.  165 

The  nature  of  the  fatty  acids  is  not  yet  determined.  It 
is  possible  that  they  differ  with  different  specimens.  They 
seem  to  be  unsaturated  acids  of  the  C16  or  Qg  series. 

Choline  or  trimethyl-/3-hydroxy-ethyl-ammoniurn  hy- 
droxide is  of  considerable  interest,  since  it  is  closely 
related  chemically  to  muscarine,  a  very  poisonous  base 
obtained  from  certain  fungi.  In  fact,  pseudo-muscarine, 
which  somewhat  resembles  muscarine,  has  recently  been 
shewn  to  be  the  nitrous  acid  ester  of  choline.  Our  present 
knowledge  concerning  choline  and  related  substances  will 
be  found  in  Barger's  "  The  Simpler  Natural  Bases  " 
(Longmans,  Green  and  Co.,  1914). 

Kephalin  differs  from  lecithin  in  being  insoluble  in 
alcohol.  Chemically,  it  differs  in  that  the  base  united  to 
the  phosphoric  acid  radicle  is  not  choline,  but  oxyethyla- 
mine,  NH2.CH2.CH2OH. 

The  preparation  of  lecithin  is  described  by  Maclean  in 
his  monograph. 

The  Galactolipins  or  Cerebrosides. 

These  compounds  do  not  contain  phosphorus.  Their 
name  is  derived  from  the  fact  that  they  yield  galactose  on 
hydrolysis,  and  are  particularly  abundant  in  the  brain, 
though  they  are  found  elsewhere  in  the  body.  Two  mem- 
bers of  this  class  have  been  described,  Phrenosin  and 
Kerasin.  Their  constitution  can  be  represented  as  follows  : 


Phrenosin 


Phrenosinic    acid,    C^  Hgo  O3,  an  a-hydroxy 

fatty  acid. 
Galactose. 
Sphingosine,  C^Hgg  NO2,  a  base. 


(Lignoceric  acid, 
Kerasin      \  Galactose. 

{Sphingosine. 


CHAPTER   VII. 
THE   CHEMISTRY   OF   SOME   FOODS. 

A.    Milk. 

The  composition  of  milk  differs  considerably  in  different 
animals.  The  percentage  composition  of  average  samples 
of  human  and  cow's  milk  is  as  follows  :— 

Protein.        Fat.        Lactose.       Salts. 
Human..          ..          i  '2  27  6*5  o'2 

Cow's      ..          ..          3*4  4'°  4*5  °*7 

Other  differences  are  that  in  cow's  milk  the  proportion  of 
casein  to  lactalbumin  is  about  6  to  i ,  compared  with  2  to  i 
in  human  milk. 

Casein,  the  chief  protein  of  milk,  is  a  phospho-protein. 
Like  all  proteins?  it  is  an  ampholyte,  but  it  differs  from  the 
majority  of  proteins  in  having  marked  acid  characters. 
The  iso-electric  point  of  casein  (see  pages  1 1  and  32)  is 
about  PH  =  4*6.  At  this  reaction  it  has  its  minimum 
solubility.  In  solutions  alkaline  to  this  it  forms  salts  with 
bases  ;  in  solutions  acid  to  this  it  forms  salts  with  acids. 
These  salts  are  more  or  less  soluble  in  water.  So  it  can 
be  stated  that  casein  is  insoluble  in  water  and  dilute  acids, 
but  soluble  in  alkalies  and  strong  acids.  Since  the  reaction 
of  milk  is  about  PH  =  7  it  follows  that  the  casein  is  held  in 
solution  as  a  salt  with  a  base.  The  base  is  probably 
calcium,  though  it  is  possible  that  a  complex  with  a  phos- 
phate is  the  condition  in  which  the  casein  exists  naturally 
in  untreated  milk. 

Casein  seems  to  have  a  molecular  weight  of  about 
8900.  It  is  readily  hydrolysed  by  alkalies  and  by  proteo- 
lytic  enzymes  into  two  molecules  of  paracasein,  which  has  a 


CH.   VII.]  MILK.  167 

molecular  weight  of  4450.  The'calcium  salts  of  paracasein 
are  very  insoluble  in  solutions  which  have  a  reaction  between 
PH  =  4  and  PH  =  7.  It  therefore  follows  that  if  casein  be 
hydrolysed  to  paracasein  in  the  presence  of  soluble  calcium 
salts  and  the  reaction  be  between  the  stated  limits,  then  the 
paracasein  will  be  precipitated  as  an  insoluble  calcium  salt. 
This  is  the  probable  explanation  of  the  well-known  phe- 
nomenon of  the  clotting  of  milk.  Casein  is  not  coagulated 
on  boiling.  But  when  milk  is  boiled  a  skin  forms  on  the 
surface.  A  similar  skin  forms  whenever  a  protein  solution 
mixed  with  an  emulsion  of  a  fat  is  heated.  The  skin  con- 
tains protein  mixed  with  fat.  If  it  be  removed,  another 
skin  immediately  forms. 

183.  Examine  a  drop  of  fresh  cow's  milk  under  the  micro- 
scope, using  a  high  power.   Notice  the  highly-refractive  fat  globules 
of  varying  size,  the  smallest  exhibiting  the  peculiar  vibration  known 
as  Brownian  movement. 

184.  Take  the  specific  gravity  of  milk  with   a  lactometer. 
It  varies  between  1028  and  1034. 

NOTE. — When  the  milk  is  skimmed  the  specific  gravity  rises  to  1037, 
owing  to  the  removal  of  the  fat  which  has  a  low  specific  gravity.  Dilution 
with  water  lowers  the  specific  gravity. 

185.  Take  the  reaction  of  milk  by  placing  drops  on  pieces 
of  red  and  blue  litmus  paper  and  then  washing  off  with  distilled 
water.     The  blue  paper  is  usually  turned  red  and  the  red  paper 
blue,  i.e.  the  milk  is  amphoteric  in  reaction. 


Casein. 

186.  Take  5  cc.  of  milk  in  a  test-tube  and  dilute  with  distilled 
water  until  the  tube  is  nearly  full.      Add  three  drops  of  strong  acetic 
acid  and  mix  thoroughly.     A  flocculent  precipitate  of  casein  is 
formed,  which  mechanically  carries  the  fat  down  with  it. 

187.  Repeat  the  above  experiment  but  add  5  cc.  of  the  strong 
acetic  acid.     Usually  no  precipitate  or  only  a  slight  one  is  obtained, 
the  casein  being  soluble  in  the  excess  of  acid. 


168  THE   CHEMISTRY   OF   SOME   FOODS.  [CH.   VII. 

iSS.  To  20  cc.  of  milk  in  a  100  cc.  measuring  cylinder  add 
65  cc.  of  distilled  water  and  15  cc.  of  i  per  cent,  acetic  acid.  Mix 
thoroughly  and  allow  to  stand  for  about  5  minutes.  Mix  again  and 
filter  through  a  pleated  paper.  The  filtrate  may  have  to  be  passed 
through  the  paper  again,  but  can  usually  be  obtained  perfectly  clear. 
Label  the  filtrate  A. 

189.  Treat  some  of  the  precipitate  obtained  in  the  previous 
exercise  with  a  little  water  and  about  i  cc.  of  2  per  cent,  sodium 
carbonate.     Shake  vigorously  in  a  test-tube.     A  milky  suspension 
of  fat  in  an  alkaline  solution  of  casein  is  obtained. 

190.  To  a  portion  of  this  suspension  cautiously  add  acetic 
acid.    The  casein  is  reprecipitated,  when  the  solution  is  definitely 
acid  to  litmus. 

191.  Transfer  some  of  the  precipitate  obtained  in  Ex.  188  or 
in  Ex.  190  to  a  test-tube.     Add  about  2  cc.  of  "  glyoxylic  reagent  " 
(Ex.  23),  and  then  2  cc.  of  pure  sulphuric  acid.     Mix  by  gentle 
agitation.     As  the  casein  dissolves  in  the  hot  acid  a  fine  purple 
glyoxylic  reaction  is  developed,  due  to  the  presence  of  tryptophane 
in  casein. 

192.  Heat  another  portion  of  the  precipitate  with  Millon's 
reagent  (Ex.  22).     The  precipitate  turns  brick  red,  owing  to  the 
presence  of  tyrosine  in  casein. 

193.  Squeeze  the  remainder  of  the  precipitate  obtained   in 
Ex.  188  between  filter  paper  to  express  as  much  fluid  as  possible. 
Reserve  a  portion  for  Ex.  194. 

Place  a  piece  about  the  size  of  a  pea  in  a  dry  test-tube  and  add 
10  drops  of  pure  sulphuric  acid.  Heat  over  a  small  flame  until  the 
mass  charrs.  Then  cautiously  add  one  drop  of  pure  nitric  acid, 
taking  care  that  an  explosive  reaction  does  not  endanger  yourself  or 
your  neighbours.  Heat  again  over  the  flame  until  white  sulphuric 
fumes  appear  in  the  tube.  (Unless  the  solution  is  heated  until  it 
fumes  in  the  tube  the  temperature  will  not  rise  sufficiently  for  the 
complete  and  rapid  oxidation  of  the  organic  material.)  If  the 
solution  again  charrs  add  another  drop  of  nitric  acid  with  the  same 


CH.    VII.]  MILK.  169 

precautions.  This  process  must  be  repeated  until  the  solution  can 
be  heated  till  sulphuric  fumes  are  found  without  the  solution  charr- 
ing. If  too  much  of  the  substance  has  been  taken  and  a  black  pasty 
mass  is  formed,  it  is  necessary  to  add  a  few  more  drops  of  sulphuric 
acid.  Allow  the  yellow  solution  to  cool,  and  then  add  about  5  cc.  of 
distilled  water.  Add  strong  ammonia,  drop  by  drop,  until  the 
reaction  is  just  alkaline,  cooling  under  the  tap  during  the  addition. 
An  alkaline  reaction  is  generally  indicated  by  the  sudden  increase 
in  the  colour  of  the  solution.  Add  a  single  drop  of  nitric  acid  to 
make  the  solution  acid.  Add  about  a  half  volume  of  ammonium 
molybdate  solution  and  boil.  A  yellow  precipitate  of  ammonium 
phospho-molybdate  indicates  the  presence  of  phosphorus  in  the 
casein.  This  is  originally  present  in  organic  combination,  but  has 
been  oxidised  by  the  above  method  (Neumann's)  into  inorganic 
phosphoric  acid. 

194.  Treat  5  cc.  of  milk  with  5  cc.  of  saturated  ammonium 
sulphate  solution.  The  casein  is  precipitated,  entangling  the  fat 
with  it.  Filter,  labelling  the  nitrate  B.  Treat  the  precipitate  with 
water.  The  casein  dissolves.  Treat  this  cloudy  solution  with 
acetic  acid.  The  casein  is  precipitated. 

NOTE. — The  casein  dissolves  in  water  because  it  is  precipitated  as  a  salt 
by  ammonium  sulphate. 


Fats. 

195.  Transfer  the  remainder  of  the  precipitate  obtained  in 
Ex.  188  to  a  dry  test-tube.     Shake  it  vigorously  with  5  cc.  of  ether. 
Pipette  off  the  ethereal  solution.     Heat  an  evaporating  basin  by 
placing  it  on  a  boiling  water  bath.     Turn  out  the  flame  and  then  add 
the  ethereal  solution  to  the  dish.    The  ether  evaporates  rapidly  and 
leaves  a  small  amount  of  fatty  residue.     Wipe  the  dish  round  with  a 
piece  of  writing-paper.    A  translucent  grease  spot  is  formed. 

196.  Estimation  of  fat  in  milk  by  Meig's  method. 

To  10  cc.  of  milk  in  a  100  cc.  glass-stoppered  cylinder  add  20  cc. 
•of  distilled  water  and  20  cc.  of  ether.  Shake  for  5  minutes.  Add 
20  cc.  of  95  to  98  per  cent,  alcohol  and  shake  again  for  5  minutes. 


170 


THE   CHEMISTRY   OF    SOME   FOODS. 


[CH.   VII, 


Allow  to  stand  until  the  mixture  has  separated  into  two  layers. 
Remove  the  upper  layer  by  the  special  pipette  shewn  in  fig.  28, 

collecting  it  in  a  glass  evaporating 
basin  that  has  been  weighed.  Add 
5  cc.  of  ether  in  such  a  way  as  to 
wash  down  the  sides  of  the  cylinder. 
Remove  this  as  before.  Repeat  the 
washing  with  5  cc.  of  ether  four 
more  times.  Evaporate  the  mixed 
ethereal  solution  to  dryness  on  a 
water  bath  or  a  special  electric 
heater.  Place  the  dish  in  a  desic- 
cator over  sulphuric  acid  until  its 
weight  is  constant. 

Important  Note. — The  greatest  care 
is  necessary  when  evaporating  off  ether 
in  open  basins.  It  is  advisable  to  place 
the  dish  on  a  boiling  water  bath,  the 
flame  having  just  previously  been  turned 
out.  No  other  flame  should  be  in  the 
vicinity.  When  the  evaporation  becomes 
slow,  remove  the  dish,  reboil  the  water, 
turn  out  the  flame  and  then  replace  the 
dish.  It  is  safer  and  more  convenient  to 
use  an  electric  heater. 


Fig.  28.  Pipette  arranged  to 
remove  the  upper  layer  in 
Meig's  method  of  fat  ex- 
traction. The  end  of  the 
tube  A  is  placed  just  above 
C,  the  surface  of  the  division 
between  the  two  layers .  The 
upper  layer  is  then  forced 
out  through  A  by  blowing  air 
into  the  space  above  B. 


Lactalbumin. 

197.  Boil  the  nitrate  B,  ob- 
tained in  Ex.  194.  A  coagulum  of 
lactalbumin  is  obtained.  (See  note 
to  Ex.  37-) 


198.  Examine  the  filtrate  A,  obtained  in  Ex.  188.  Add  a  drop 
or  two  of  litmus,  and  note  that  the  reaction  is  distinctly  acid.  Boil, 
and  whilst  boiling  add  2  per  cent,  sodium  carbonate,  drop  by  drop,, 
until  the  reaction  is  only  faintly  acid.  A  coagulum  of  lactalbumin 
is  formed.  Filter  this  off  and  reserve  the  filtrate  (C). 

NOTE. — On  boiling  the  acid  solution,  the  lactalbumin  is  converted  to- 
metaprotein  (Ex.  29).  On  neutralising  the  metaproteins  are  precipitated,  and 
since  the  solution  is  boiling  they  are  coagulated  (Ex.  47).  The  solution  must 
not  be  made  alkaline,  for  this  would  cause  the  earthy  phosphates  to  be  pre- 
cipitated. 


CH.    VII.]  MILK.  171 

Lactose. 

199.  Boil  a  small  portion  of  filtrate  C  with  a  little  Fehling's 
solution.     A  well-marked  reduction  is  obtained,  due  to  the  presence 
of  a  reducing  sugar. 

200.  A.     Measure  2  cc.  of  filtrate  C  into  a  test-tube.     Add  3 
drops  of  glycerol ;  add  10  drops  of  20  per  cent,  copper  sulphate  by 
means  of  a  Dreyer's  dropping  pipette  (fig.  5),  and  then  2  cc.  of  20 
per  cent,  sodium  hydroxide.     Boil  the  mixture  and  keep  it  boiling 
for  one  minute.     Allow  the  tube  to  stand.     If  the  supernatant  fluid 
is  blue,  repeat  the  experiment  with  less  copper.   If  the  fluid  is  yellow, 
repeat  with  more  copper.      The  approximate  amount  of  copper 
that  is  reduced  by  the  sugar  in  2  cc.  of  the  fluid  is  thus  found. 

B.  Measure  2  cc.  of  filtrate  C  into  a  test-tube  and  add  0-5  cc. 
of  pure  concentrated  hydrochloric  acid.  Boil  gently  over  a  small 
flame  for  two  minutes.  Cool  and  add  14  drops  of  the  copper 
sulphate,  and  3  drops  of  glycerol.  Neutralise  by  means  of  20  per 
cent,  sodium  hydroxide,  the  neutral  point  being  indicated  by  the 
appearance  of  a  grey  precipitate.  Now  add  a  further  2  cc.  of  the 
sodium  hydroxide  and  boil  for  one  minute.  The  whole  of  the  copper 
is  usually  reduced.  The  increase  in  the  reducing  power  after  boiling 
with  hydrochloric  acid  demonstrates  that  the  sugar  present  in  milk 
is  not  glucose  (see  Ex.  103). 

201.  To  5  cc.  of  Barfoed's  solution  add  i  cc.  of  filtrate  C  and 
repeat  Ex.  101.    A  reduction  is  not  obtained.    This  experiment,  in 
conjunction  with  the  previous  one,  indicates  that  the  sugar  present 
is  lactose  or  maltose. 

202.  Concentrate  about  25  cc.  of  filtrate  C  to  about  10  cc.  on 
the  water  bath.    Transfer  this  to  a  test-tube.    Add  i  cc.  of  strong 
acetic  acid  and  proceed  as  in  Ex.  109.     Allow  the  yellow  solution 
that  is  obtained  to  cool  slowly.     A  yellow  precipitate  of  lactosazone 
appears.   Filter  through  a  small  paper,  and  suspend  in  about  4  cc. 
of  water.     Boil.     The  precipitate  dissolves.     Allow  to  cool  slowly 
and  examine  the  precipitate  under  the  microscope.     Lactosazone 
usually  crystallises  in  solid  ovoid  clumps  with  a  projecting  fringe  of 
fine  needles.     ("  Hedge-hog  "  crystals.) 


172  THE   CHEMISTRY   OF   SOME    FOODS.  [cH.    VII. 

203.  The  estimation  of  lactose  in  milk  by  the  method  of 
Folin  and  Denis.* 

Principle.  Proteins  do  not  interfere  with  the  method  of 
estimation  of  sugar  devised  by  Folin  and  McEllroy  (Ex.  161). 

Method.  Dilute  the  milk  i :  4  (25  cc.  to  100  cc.)  for  cow's 
milk  and  i :  5  (5  cc.  to  25  cc.)  for  mother's  milk.  Fill  the  special 
burette  with  the  diluted  milk  (or  use  a  burette  of  the  usual 
pattern). 

Into  a  large  tube  place  5  grams,  of  the  phosphate  powder,  5  cc. 
of  the  6  per  cent,  copper  sulphate,  shake  and  boil.  Run  in  about 
3-4  cc.  of  the  diluted  milk  and  boil  gently  for  4  minutes.  Complete 
the  titration  as  described  in  Ex.  161. 

~  7    7  ..          4-04  x  dilution 

Calculation.  -  =  anhydrous  lactose  per  cent, 

volume  required 

NOTE. — The  average  amount  of  lactose  in  cow's  milk  is  4-5  per  cent.  So 
about  3-6  cc.  of  a  i  in  4  dilution  of  normal  cow's  milk  is  required. 


Inorganic  constituents. 

204.  Treat  the  remainder  of  nitrate  C  with  two  or  three  drops 
of  strong  ammonia  and  boil.     A  slight  gelatinous  precipitate  of 
calcium  phosphate  is  produced.     Filter  through  a  small  paper. 
Boil  4  cc.  of  water,  to  which  has  been  added  i  cc.  of  strong  acetic 
acid.     Pour  the  hot  solution  on  to  the  filter  paper,  and  collect  the 
filtrate  in  a  clean  tube.     To  the  filtrate  add  a  solution  of  potassium 
oxalate.     A  white  precipitate  of  calcium  oxalate  is  formed.     Treat 
with  i  cc.  of  nitric  acid.     The  precipitate  dissolves.     Add  a  few 
cc.  of  ammonium  molybdate  solution  and  boil.     A  yellow  crystalline 
precipitate  is  slowly  formed,  indicating  the  presence  of  phosphates 
in  milk. 

C.    Cheese. 

205.  Shake  some  grated  cheese  in  a  dry  test-tube  with  ether, 
and  examine  the  ethereal  solution  for  fat  as  in  Ex.  195.     Fats  and 
fatty  acids  are  present  in  considerable  quantity. 

*  Journal  of  Biological  Chemistry,  xxxiii.,  p.  521    (1918). 


CH.    VII.]  POTATOES   AND   FLOUR.  173 

206.  Pound  the  residue  from  the  above  in  a  mortar  with  a 
2  per  cent,  solution  of  sodium  carbonate  and  filter.     Acidify  the 
filtrate  with  acetic  acid.     A  precipitate  of  casein  is  formed,  which 
is  soluble  in  excess  of  acid.   To  the  filtrate  from  this  apply  the  usual 
protein  colour  reactions :  they  are  usually  all  obtained  owing  to 
the  presence  of  proteoses,  peptones  and  various  animo-acids. 

D.    Potatoes. 

207.  Scrape  the  clean  surface  of  half  a  potato  with   a  pen- 
knife, keeping  the  scrapings  as  fine  as  possible.    Place  the  scrapings 
in  a  beaker  of  water,  stir  well,  and  strain  through  fine  muslin  into- 
another  beaker.     Allow  this  to  stand  for  a  few  minutes  and  then 
note  the  white  deposit  of  starch.     Pour  off  the  supernatant  fluid 
and  reserve  it  for  the  next  exercise.     Fill  the  beaker  containing 
the  starch  with  water,  stir  well,  and  again  allow  the  starch  to  settle. 
By  repeating  this  process  of  lixiviation  the  starch  can  be  obtained 
quite  pure.    Examine  a  little  microscopically  and  note  the  charac- 
teristic form  of  the  grains  (see  Ex.  132) .     Heat  a  little  with  \vater, 
cool,  and  add  iodine.     A  deep  blue  colour  is  obtained. 

208.  Filter  the  fluid  A,  and  test  portions  of  the  filtrate  for 
proteins  by  the  usual  colour  tests.     Only  small  quantities  of  protein, 
are  found  to  be  present,  the  most  marked  reaction  being  Millon's. 

E.     Flour. 

White  flour  from  the  endosperm  of  wheat  grains 
contains  70  to  75  per  cent,  of  starch,  about  8  per  cent,  of 
protein  and  about  i  per  cent,  of  fat.  The  proteins  are 
gliadin  (soluble  in  70  to  80  per  cent,  alcohol),  and  glutelin 
(soluble  in  alkali).  When  treated  with  water  these  two 
proteins  form  a  sticky  mass  called  gluten,  the  viscidity 
being  due  to  the  gliadin.  Thus  grains  poor  in  gliadin,  as 
rice  and  oats,  do  not  form  dough  when  mixed  with  water. 

Flour  only  contains  glucose  if  germination  has  taken 
place  before  milling. 

Whole  flour  is  obtained  from  the  whole  of  the  grain, 
except  the  outer  husk  and  outer  part  of  the  bran.  It  is 


174  THE   CHEMISTRY    OF    SOME    FOODS.  [CH.    VII. 

possible  that  it  contains  something  essential  to  growth 
and  general  nourishment.  It  is  not  quite  so  digestible  as 
white  flour.  The  bran  in  it  stimulates  the  intestine  and 
so  acts  as  a  mild  laxative. 

209.  Mix  some  wheat  flour  with  a  little  water  to  form  a  stiff 
dough.     Allow  this  to  stand  for  a  short  while,  preferably  at  37°  C. 

Wrap  a  piece,  the  size  of  a  chestnut,  in  muslin,  and  knead  it 
for  a  few  minutes  in  a  basin  of  water ;  pour  the  suspension  into  a 
beaker,  and  note  the  white  deposit  of  starch  grains  that  settles  down 
on  standing.  Examine  this  microscopically,  noting  that  the  grains 
differ  considerably  from  those  of  potato-starch  in  being  smaller, 
circular,  and  with  a  central  hilum.  Make  a  drawing  of  the  grains. 
Boil  a  little  with  water,  cool,  and  add  a  drop  of  iodine.  The  deep 
blue  starch  reaction  is  obtained. 

210.  Knead  the  dough  thoroughly  under  the  tap  until  no  more 
starch  comes  through  the  muslin.     A  yellowish,  sticky  mass,  known 
as  gluten,  is  left  behind.     Test  portions  of  this  by  the  usual  protein 
colour  reactions :  they  are  all  obtained,  gluten  being  a  protein. 

F.    Bread. 

The  dough  formed  by  adding  water  to  flour  is  imper- 
vious to  the  digestive  juices.  Before  it  can  be  used  it  has 
to  be  aerated  and  the  gluten  rendered  porous. 

A  pure  culture  of  yeast  is  mixed  with  warm  water, 
flour  and  salt.  The  dough  thus  formed  is  thoroughly 
kneaded,  and  the  mass  kept  warm  for  some  hours.  During 
this  time  the  yeast  cells  multiply  and  convert  some  of  the 
starch  into  glucose  and  this  into  alcohol  and  CO2.  Also 
the  ferment  of  the  flour  called  diastase  becomes  active  and 
converts  some  of  the  starch  into  glucose.  More  flour  is 
added  and  the  process  allowed  to  proceed  for  some  hours 
longer.  The  gas  formed  causes  the  mass  to  rise.  The 
dough  is  weighed  out  into  loaves,  which  after  being  allowed 
to  rise  once  more  for  a  certain  time  are  heated  to  about 
232°  C.  for  an  hour  and  a  half.  The  heat  kills  the  yeast, 
expands  the  gas  bubbles,  and  causes  the  outer  part  of  the 


CH.    VII.]  BREAD  175 

dough  to  become  hardened  by  coagulating  the  proteins. 
It  also  converts  starch  into  soluble  starch  and  dextrin,  thus 
forming  the  crust.  The  brown  appearance  of  this  is  due 
to  the  conversion  of  glucose  into  caramel. 

211.  Take  a  piece  of  the  crumb  of  a  stale  .white  loaf,  rub  it 
up  finely  and  pound  with  cold  water  in  a  mortar.     Strain  and 
squeeze  through  muslin.     A  white  fluid  is  obtained  containing 
wheat  starch  grains.     Filter  the  fluid.     To  a  portion  of  the  filtrate 
add  a  little  Fehling's  solution  and  boil :  a  well-marked  reduction 
occurs  due  to  the  presence  of  glucose.     To  another  portion  add 
iodine  :  a  purple  colour  is  produced,  showing  the  presence  of  erythro- 
dextrin.     If  very  dilute  iodine  be  cautiously  added,  a  blue  colour 
is  produced  at  first,  showing  that  a  small  amount  of  soluble  starch  is 
present. 

Boil  a  small  amount  of  the  residue  of  the  bread  with  water  in  a 
beaker,  strain  through  muslin  and  filter.  Cool  and  test  the  filtrate 
for  starch  and  dextrin.  (Ex.  145  to  147.) 

212.  Repeat  the  above  exercise,  using  the  crust  of  bread 
instead  of  the  crumb.     Note  that  glucose  is  absent  or  present  in 
traces  only :  dextrin  and  starch  are  present,  a  considerable  portion 
of  the  latter  existing  as  soluble  starch  and  being  present  in  the  cold 
water  extract. 


G.    Meat  (Muscle). 

The  most   important   constituents   of  living  striated 
muscle  are  : — 

Proteins.     Myosinogen    and    Paramyosinogen. 

Pigment.     Myohaematin. 

Fat. 

Nitrogenous  extractives.    Creatine. 

Hypoxanthine. 

Xanthine. 

Carnosine. 


176  THE    CHEMISTRY    OF    SOME    FOODS.  [CH.    VIK 

Non-nitrogenous  extractives.     Glycogen. 

Sarcolactic  acid. 

Inorganic.      Water. 

Salts,  chiefly  potassium  and  magnesium 
phosphates. 

The  proteins  of  living  muscle  are  mainly  myosinogen 
(80  per  cent.)  and  paramyosinogen  (20  per  cent.).  The 
former  is  an  albumin,  coagulating  at  57°  C.  The  latter  is 
a  globulin,  coagulating  at  47°  C. 

On  standing  or  on  treatment  with  dilute  acids  they 
are  converted  into  myosin  the  protein  of  dead  muscle.  In 
this  transformation,  myosinogen  passes  through  an  inter- 
mediate stage  of  soluble  myosin  which  coagulates  at  40°  C. 

Myosinogen.  Paramyosinogen. 

Soluble  myosin. 
Myosin. 

213.  Preparation  o!  fresh  muscle   extract.       A   rabbit   is 
killed,  a  cannula  fixed  into  the  aorta  and  an  opening  made  in  the 
right  auricle.     The  vessels  are  then  washed  free  from  blood  with 
0-9  per  cent,  sodium  chloride.   The  muscles  of  the  limbs  are  removed, 
rapidly  minced  and  treated  with  ice-cold  5  per  cent,  magnesium 
sulphate,  and  the  mixture  left  in  the  ice  chest  for  about  24  hours. 
The  extract  is  filtered  and  the  following  tests  performed  with  it : 

214.  Take  the  reaction  to  litmus.     It  is  generally  neutral. 

215.  Dilute  a  small  portion  with  four  volumes  of  distilled 
water  and  leave  the  tube  in  the  water  bath  at  37°  C.  for  some  time, 
A  clot  of  myosin  forms,  leaving  muscle  serum. 

216.  Take  the  reaction  of  the  muscle  serum  to  litmus.     It  is 
distinctly  acid,  due  to  the  production  of  sarcolactic  acid. 

217.  Add  some  acetic  acid  to  another  portion  of  the  extract. 
A  precipitate  of  myosin  occurs  immediately. 


CH.   VII.]  MEAT.  177 

218.  Take  5  cc.  of  the  extract  in  a  test-tube :  place  the  tube 
in  a  beaker  of  water,  supporting  it  by  a  clamp  so  that  it  does  not 
touch  the  bottom  of  the  beaker.    Heat  the  water  with  a  Bunsen 
flame  and  note  the  temperature  in  the  tube    at  which  distinct 
coagulation  occurs.     It  is  usually  at  about  47°  C.     Filter  off  the 
coagulum  of  paramyosinogen  and  heat  again.     Another  and  larger 
coagulum  of  myosinogen  occurs  at  57°  C. 

219.  Preparation  of  Myosin.     Fresh  veal  is  finely  minced  in  a 
machine,  stirred  with  a  large  volume  of  water  for  a  quarter  of  an 
hour,  strained  through  muslin,  and  the  washing  process  repeated 
once  more.     In  this  way  certain  proteins  and  other  substances 
soluble  in  water  are  removed.     The  veal  is  now  collected  on  muslin, 
squeezed  to  remove  the  water,  ground  with  sand,  and  extracted 
with  five  times  its  volume  of  10  per  cent,  ammonium  chloride  for 
several  hours  at  room  temperature.     The  extract  is  filtered  through 
muslin,  linen,  and  then  coarse  filter  paper.     In  this  way  a  crude, 
viscid  solution  of  myosin  is  obtained. 

220.  Boil  a  portion  of  the  solution.     A  heavy  coagulum  is 
formed.     Wash  the  coagulum  and  on  it  perform  the  protein  colour 
reactions.     They  are  all  obtained. 

221.  Pour  100  cc.  into  a  litre  of  water  contained  in  a  tall 
cylinder ;  mix  well,  and  note  the  precipitation  of  myosin,  due  to  the 
reduction  in  the  concentration  of  salts. 

Allow  this  to  settle,  and  then  pour  or  pipette  off  as  much  of  the 
supernatant  fluid  as  possible.  A  suspension  of  myosin  in  dilute 
ammonium  chloride  is  thus  obtained  for  the  next  three  experiments. 

NOTE. — If  this  suspension  be  allowed  to  stand  it  slowly  becomes  con- 
verted into  an  insoluble  variety. 

222.  To  a  portion  add  a  saturated  solution  of  common  salt, 
drop    by  drop.     The  precipitate  dissolves.     Add  solid  NaCl  to 
saturation :  the  myosin  is  reprecipitated. 

223.  To  a  portion  add  saturated  ammonium  sulphate  till  the 
precipitate  just  dissolves.     Now  add  an  equal  bulk  of  saturated 
ammonium  sulphate.     The  myosin  is  reprecipitated. 


178  THE   CHEMISTRY    OF    SOME    FOODS.  [CH.   VII. 

224.  Dissolve  in  a  little  ammonium  sulphate  and  take  the 
temperature  at  which  the  myosin  coagulates.     It  coagulates  at 
about  57°  C.  (see  Ex.  218). 

Creatine. — This  is  the  most  abundant  nitrogenous 
extractive  in  muscle,  being  present  to  the  extent  of  about 
0*4  per  cent.  Chemically  it  is  methyl-guanidine-acetic 
acid. 

NH  CH3 

II       I 
C  — N— CH2 

NH2        COOH 

On  hydrolysis  with  baryta  water  it  is  converted  into 
urea  and  sarcosine  (methyl  glycine). 

NH  CH3  NH2    CH3 

II         I  I 

NH2.C N.CH2.COOH+H20     =     NH2.CO  +  NH.CH2.COOH. 

Urea.  Sacrosine. 

On  being  boiled  with  mineral  acids  it  is  dehydrated 
to  creatinine. 

NH  CH3 

II        I 
C  —  N  —  CH2 

NH-      -CO 

Creatinine  is  found  in  normal  human  urine,  but 
creatine  only  under  abnormal  conditions. 

225.  Separation  of  creatine  from  meat   extract.      Dissolve 
10  grams,  of  commercial  meat  extract  in  200  cc.  of  water.     Add 
slowly  a  saturated  solution  of  lead  acetate  till  no  further  precipitate 
is  formed,  carefully  avoiding  an  excess.     This  is  best  done  by 
filtering  samples  and  testing  them  with  lead  acetate.     Filter  off  the 
precipitate  of  proteins  and  phosphates.     Warm  the  nitrate  and 
decompose  the  soluble  lead  compounds  by  means  of  a  stream  of 
sulphuretted  hydrogen.     Warm  and  filter  off  the  precipitate  of 


CH.   VII.]  MEAT.  179 

lead  sulphide.  Evaporate  the  filtrate,  filtering  off  any  sulphur  or 
sulphide  that  may  be  deposited.  Continue  the  evaporation  till  a 
syrup  is  obtained.  Allow  this  to  stand  in  the  ice  chest  for  two  or 
three  days.  Creatine  separates  out,  mostly  as  oblique  rhombic 
crystals.  Examine  a  few  under  the  microscope.  Treat  the  syrup 
with  200  cc.  of  88  per  cent,  alcohol,  stir  thoroughly  with  a  glass 
rod  and  filter  through  a  small  paper.  The  creatine  remains  on  the 
paper,  the  alcoholic  filtrate  containing  the  purine  bases. 

NOTE. — Many  specimens  of  commercial  meat  extract  contain  creatinine 
as  well  as  creatine.  Rabbit's  muscle  is  the  best  source  of  pure  creatine.  The 
muscle  is  finely  minced,  extracted  with  boiling  water,  the  proteins  removed 
by  boiling  and  adjusting  the  reaction.  The  filtrate  is  worked  up  as  described 
above. 

226.  Conversion  of  creatine  into   creatinine.     Dissolve  the 
creatine  in  about  30  cc.  of  hot  water  and  divide  the  solution  into  two 
equal  portions,  A  and  B.     Treat  B  with  an  equal  volume  of  normal 
HC1  and  heat  on  a  boiling  water  bath  in  a  flask  fitted  with  a  cork 
and  long  glass  tube  (to  act  as  an  air  condenser)  for  three  to  five  hours. 
The  creatine  is  converted  into  creatinine.     Neutralise  the  solution 
with  caustic  soda. 

Test  A  and  B  for  creatinine  by  the  following  tests: 

227.  Jaffe's  test  for  creatinine.    Treat  10  cc.  of  the  solution 
with  15  cc.  of  saturated  picric  acid  solution  and  5  cc.  of  10  per 
cent,  caustic  soda.     Allow  the  mixture  to  stand  for  5  minutes  and 
dilute  to  200  cc.     A  deep  orange  colour  appears  in  B  due  to  the 
formation  of  picramic  acid  from  creatinine.     The  creatine  in  A  gives 
no  colour. 

228.  Weyl's  test  for  creatinine.    Treat  5  cc.  with  a  few  drops 
of  a  freshly  prepared  sodium  nitroprusside  and  make  the  solution 
just  alkaline  with  sodium  hydroxide.     A  ruby-red  colour  appears. 
Boil.     The   solution   turns   yellow.     Acidify   with   an   excess   of 
acetic  acid  and  heat.     A  green  tint  appears,  and  a  blue  deposit  of 
Prussian  blue  may  result  on  standing. 

Purine  bases.  These  compounds  are  interesting  because 
of  their  chemical  relationship  to  uric  acid.  This  relation- 
ship is  shewn  by  the  formulae  given  on  p.  62. 


180  THE    CHEMISTRY   OF    SOME    FOODS.  [CH.   VII. 

The  purine  bases  found  in  meat  extracts  are  chiefly 
hypoxanthine  and  xanthine.  They  can  be  obtained  from 
the  alcoholic  solution  obtained  in  Ex.  166,  by  evaporating 
off  the  alcohol,  adding  ammonia  and  precipitating  with 
ammoniacal  silver  nitrate. 

Sarcolactic  acid  is  ctoro-a-hydroxy-propionic  acid. 

OH 

CH3.CH.COOH. 

The  lactic  acid  found  in  muscle  is  d-lactic.  That 
formed  by  the  fermentation  of  lactose  and  other  carbo- 
hydrates is  generally  ^/-lactic.  Certain  bacteria,  however, 
produce  /-lactic  acid  (see  p.  151). 

Sarcolactic  acid  is  present  to  a  very  small  extent  in 
fresh  living  muscle.  The  amount  increases  rapidly  in 
fatigue,  especially  in  the  absence  of  a  proper  supply  of 
oxygen.  On  leaving  a  fatigued  muscle  in  an  atmosphere 
of  oxygen,  the  amount  of  lactic  acid  decreases. 

There  is  a  considerable  production  of  lactic  acid  at  the 
onset  of  rigor  mortis.  But  if  a  fresh  muscle  be  suddenly 
coagulated  by  dropping  it  into  boiling  water,  there  is  no 
such  marked  production  of  the  acid. 

It  is  probable  that  the  lactic  acid  appearing  in  fatigue 
and  in  rigor  arises  through  the  decomposition  of  some 
carbohydrate  material  in  the  muscle,  but  this  has  not  been 
definitely  established. 

Sarcolactic  acid  is  a  liquid,  soluble  in  water,  alcohol 
and  ether.  It  forms  a  characteristic  zinc  salt,  which  is 
obtained  by  boiling  a  solution  with  excess  of  zinc  carbonate, 
filtering  and  evaporating  slowly.  The  crystals  contain 
two  molecules  of  water  of  crystallisation,  the  zinc  salt  of 
ordinary  fermentation  lactic  acid  containing  three. 

229.  Hopkins'  reaction  for  lactic  acid.  To  3  drops  of 
a  i  per  cent,  alcoholic  solution  of  lactic  acid  in  a  clean,  dry  test 
tube  add  5  cc.  of  concentrated  sulphuric  acid  and  3  drops  of  a 


CH.    VII.]  LACTIC   ACID.  181 

saturated  solution  of  copper  sulphate.  Mix  and  place  the  tube  in  a 
beaker  of  boiling  water  for  about  five  minutes.  Cool  thoroughly 
under  the  tap,  add  two  drops  of  a  0-2  per  cent,  alcoholic  solution  of 
thiophene,  and  shake.  Replace  the  tube  in  the  boiling  water  bath. 
As  the  mixture  gets  warm  a  fine  cherry-red  colour  develops. 

NOTE. — Lactic  acid  is  oxidised  in  sulphuric  acid  solution  to  formaldehyde 
and  acetaldehyde  which  react  with  thiophene  in  the  presence  of  an  excess  of 
sulphuric  acid  to  give  a  cherry-red  colour.  The  copper  sulphate  aids  this 
oxidation,  which  is  inhibited  by  water. 

230.  Uffelmann's  reaction  for  lactic  acid.    Treat  a  few  cc. 
of  Uffelmann's  reagent  with  a  few  cc.  of  a  dilute  (0-4  per  cent.) 
solution  of  lactic  acid.     The  violet  colour  is  instantly  turned  to 
a  yellow. 

NOTES. — i.  Uffelmann's  reagent  is  prepared  by  treating  a  i  per  cent, 
solution  of  phenol  (carbolic  acid)  with  very  dilute  ferric  chloride  till  the 
solution  becomes  coloured  an  amethyst-violet. 

2.  The  reaction  is  not  very  reliable,  since  other  acids  as  tartaric,  oxalic 
and  citric  give  it. 

231.  The  Formation  of  Lactic  Acid  in  Fatigue.     A  pithed 
frog  is  kept  on  ice  for  about  half-an-hour.     Remove  one  hind  limb 
and  replace  it  on  the  ice.     Expose  the  lumbar  plexus  of  the  other 
side  and  stimulate  it  electrically  by  means  of  a  strong  interrupted 
current  for  at  least  ten  minutes.     Cut  off  the  hind  limb,  strip  the 
skin  off  the  two  limbs  and  treat  the  muscles  separately  as  follows : 
Rapidly  remove  the  muscles,  grind  them  with  ice-cold  95  per  cent, 
alcohol  and  sand.     Transfer  the  mixture  to  a  beaker,  and  warm  for 
a  few  minutes  on  the  water  bath.     Filter  through  a  small  paper 
and  evaporate  to  complete  dryness  on  a  water  bath.    Treat  the 
residue  with  about  5  cc.  of  cold  water  and  rub  it  up  thoroughly 
with  a  glass  rod.     Filter  and  boil  the  filtrate  with  as  much  animal 
charcoal  as  will  lie  on  a  threepenny  piece.     Filter  and  evaporate 
the  filtrate  to  complete  dryness  on  a  water  bath.     Allow  the  residue 
to  cool  and  apply  Hopkins'  test  by  treating  the  residue  with  strong 
sulphuric  acid,  shaking  round  till  solution  is  obtained,  transferring 
to  a  dry  test-tube,  adding  three  drops  of  saturated  copper  sulphate, 
etc.     A  fine  red  colour  develops  in  the  tube  containing  the  extract 
from  the  tetanised  muscle,  but  none  or  very  little  in  the  other. 


CALIFORNIA  COlU«i 

of  CY  ' 


182 


THE    CHEMISTRY   OF   SOME    FOODS. 


CH.   VII. 


Glycogen.  The  percentage  of  glycogen  in  fresh  muscle 
varies  from  0*5  to  i  per  cent.,  so  that  the  total  amount  in 
all  the  muscles  of  the  body  may  be  greater  than  in  the  liver. 
The  muscle  glycogen  decreases  after  muscular  exercise, 
but  not  so  rapidly  as  that  in  the  liver. 

The  estimation  of  glycogen  is  described  on  p.  123. 


CHAPTER   VI I L 

THE  COMPOSITION   OF  THE  DIGESTIVE  JUICES 
AND    THE    ACTION    OF    CERTAIN    ENZYMES. 

The  digestive  enzymes  or  ferments  are  bodies  that 
have  the  power  of  accelerating  the  rate  of  hydrolysis  of 
certain  substances.  They  are  divided  into  groups  depend- 
ing on  the  nature  of  the  substance  on  which  the}^  act 
(the  so-called  substrate).  Thus  those  acting  on  starch 
are  called  amylolytic  ;  on  proteins,  proteolytic ;  on  fats, 
lipolytic,  etc.  The  enzymes  are  sometimes  named  in 
such  a  way  as  to  indicate  their  origin  and  their  action,  the 
termination  -ase  being  employed.  Thus  ptyalin,  the 
amylolytic  enzyme  of  saliva,  can  be  termed  salivary 
amylase,  to  distinguish  it  from  pancreatic  amylase  (amy- 
lopsin).  Gastric  lipase,  the  lipolytic  enzyme  of  the  gastric 
juice,  is  similarly  distinguished  from  pancreatic  lipase 
(steapsin). 

The  chemical  composition  of  the  enzymes  is  at  present 
uncertain,  owing  to  the  extreme  difficulty  of  preparing 
them  in  a  pure  state.  The  proteolytic  enzymes  are  either 
proteins,  or  compounds  so  readily  adsorbed  by  proteins 
that  it  is  impossible  to  separate  them.  The  enzymes  acting 
on  certain  of  the  carbohydrates  are  possibly  themselves 
of  a  carbohydrate  nature. 

The  properties  of  the  enzymes  as  a  class  are  as  follows  : 
they  are  soluble  in  water,  dilute  salt  solutions,  dilute 
alcohol  and  glycerol.  They  are  precipitated  by  saturation 
with  ammonium  sulphate  and  by  strong  alcohol.  They 
are  readily  carried  down  by  different  precipitates,  probably 
by  a  process  of  adsorption.  They  are  colloidal  and  non- 
diffusible.  They  are  most  active  at  a  certain  temperature, 


184  COMPOSITION   OF  THE   DIGESTIVE   JUICES.         [cH.   VIII. 

called  the  optimum  temperature,  which  is  generally  about 
45°  C.  Their  action  is  suspended  by  cooling,  but  is  com- 
pletely destroyed  by  raising  the  temperature  to  100°  C. 

The  enzymes  are  remarkably  specific  in  their  action, 
that  is,  they  act  only  on  a  particular  substance  or  on  a 
group  of  substances  having  some  similarity  in  chemical 
composition  and  configuration.  A  striking  example  of 
this  is  seen  in  the  case  of  the  glucosides  (see  page  103). 
The  enzyme  maltase  (a-glucase)  hydro lyses  a-methyl-  and 
a-ethyW-glucosides,  but  has  no  action  on  /3-methyl-  or 
/?-ethyl-d-glucosides,  or  on  any  /-glucoside  or  on  d-  or 
/-galactosides.  The  enzyme  emulsin  (/3-glucase)  acts  only 
on  /3-ethyl,  methyl  or  phenyW-glucosides.  Lactase  acts 
only  on  the  /5-galactosides.  It  is  probable  that  the  enzyme 
first  unites  with  the  substrate,  and  to  do  this  it  must  have 
a  configuration  in  space  corresponding  with  that  of  the 
substrate.  According  to  Bayliss  the  preliminary  union  of 
enzyme  with  substrate  is  a  process  of  adsorption.  Though 
adsorption  phenomena  are  probably  of  great  importance 
in  enzyme  action,  this  does  not  give  any  simple  explana- 
tion of  the  remarkable  specificity  which  is  characteristic 
of  enzyme  action,  but  not  of  adsorption. 

An  important  factor  in  the  action  of  an  enzyme  is  the 
concentration  of  hydrogen  ions  in  the  medium  in  which  it 
acts.  For  each  enzyme  there  is  a  particular  hydrogen-ion 
concentration  or  PH  at  which  the  velocity  of  reaction  is 
greatest.  This  is  the  optimum  reaction  of  that  particular 
enzyme.  The  optimum  PH  of  certain  enzymes  is  given  on 
page  23.  It  is  interesting  to  note  that  in  the  case  of 
trypsin  the  optimum  reaction  seems  to  vary  with  the 
nature  of  the  substrate,  being  PH  =  8  *o  when  acting  on 
trypsin  and  PH  =  6*7  when  acting  on  casein.*  Michaelis 
has  made  a  special  study  of  the  significance  of  the  optimum 
reaction,  and  claims  that  it  is  partly  dependent  on  the  effect 


*  The  author  has  not  been  able  to  confirm  this,  finding  pH  =  8-1  the 
optimum  reaction  for  the  hydrolysis  of  casein  by  trypsin. 


CH.   VIII.]  ENZYMES.  185 

of  the  reaction  on  the  nature  of  the  dissociation  of  the 
enzyme,  which  in  many  cases  functions  as  an  ampholyte 
(see  pages  u  and  31).  At  the  isoelectric  point  of  an 
ampholyte  it  exists  mainly  in  the  undissociated  state.  If 
the  hydrogen-ion  concentration  be  greater  than  at  the 
iso-electric  point,  the  enzyme  is  positively  charged,  that  is, 
it  exists  mainly  as  kations  :  if  the  solution  be  alkaline 
to  the  iso-electric  point,  the  enzyme  exists  mainly  as  anions, 
Michaelis  has  determined  the  iso-electric  points  of  certain 
of  the  enzymes  by  various  methods,  and  concludes  that 
maltase,  trypsin  and  erepsin  are  only  active  as  anions  ; 
pepsin  as  kations  ;  whilst  invertase  is  only  active  as  un- 
dissociated molecules.  He  makes  the  interesting  sug- 
gestion that  though  undissociated  pepsin  has  no  action  on 
ordinary  proteins,  yet  it  has  the  property  of  clotting  milk, 
that  is  a  rennetic  action  (see  p.  208). 

The  action  of  most  enzymes  is  retarded  by  the  accumu- 
lation of  the  products  of  the  reaction,  and  in  certain  cases 
the  reaction  is  reversible. 

This  is  well  seen  in  the  case  of  lipase,  which  induces 
the  following  reaction  :— 

Ethyl  butyrate  +  water  ~ >  ethyl  alcohol  +  butyric  acid. 

The  velocity  of  reaction  is  proportional  to  the  amount 
of  the  enzyme  present,  provided  that  the  amount  of  the 
enzyme  is  very  small  compared  with  that  of  the  substrate. 
If  the  amounts  of  enzyme  and  substrate  are  at  all  com- 
parable, the  laws  of  mass  action  are  followed.  But  com- 
plications are  introduced  by  the  fact  that  some  of  the 
enzyme  is  thrown  out  of  action  by  being  absorbed  by  the 
products  of  the  action. 

In  certain  cases  enzyme  action  is  dependent  on  the 
simultaneous  presence  of  two  substances.  These  are 
sometimes  called  co-ferments.  It  has  been  shewn  that 
the  zymase  that  is  responsible  for  the  alcoholic  fermenta- 
tion of  sugar  by  yeast  can  only  act  in  co-operation  with 
phosphates  and  some  substance  that  is  diffusible  and  not 


! 


186  COMPOSITION   OF  THE   DIGESTIVE    JUICES.         [cH.    VIII. 

destroyed  by  boiling.  Also  the  lipase  of  the  pancreas 
requires  the  presence  of  some  soluble,  heat-stable  sub- 
stance to  allow  it  to  act.  Bile  salts  have  this  property,  as 
has  been  seen  in  a  previous  chapter.  The  action  of  the 
enzymes  can  be  retarded  by  certain  substances.  These 
are  of  two  classes  :  paralysers  and  anti-enzymes.  The 
paralysers  are  generally  salts  of  the  heavy  metals,  which 
probably  alter  the  physical  state  of  the  colloidal  enzymes. 
The  anti-enzymes  are  of  an  organic  nature.  They  probably 
combine  with  the  enzyme  and  thus  prevent  it  from  acting 
on  the  substrate.  Examples  are  seen  in  the  case  of  the 
anti-trypsin  of  normal  serum,  of  the  intestinal  mucous 
membrane  and  of  the  tissues  of  intestinal  parasitic  worms. 

A.  Saliva. 

Saliva  is  of  value  as  a  lubricant  in  the  act  of  degluti- 
tion, and  in  some  animals  this  is  its  sole  function. 

232,  Collect  about  5  cc.  of  your  own  saliva  in  a  small  beaker. 
Test  the  reaction  with  neutral  litmus  paper :  it  is  alkaline. 

NOTE. — The  first  portion  of  saliva  collected  is  very  apt  to  be  neutral, 
or  even  slightly  acid,  probably  owing  to  bacterial  decomposition  in  the  mouth. 
But  if  the  secretion  is  free,  that  collected  later  is  invariably  alkaline. 

233.  Transfer  the  saliva  to  a  test-tube  and  add  strong  acetic 
acid.     A  stringy  precipitate  of  mucin  is  formed,  insoluble  in  excess 
of  acid.     Stir  the  mixture  vigorously  with  a  glass  rod :  the  mucin 
forms  a  clump  which  can  be  removed  by  the  rod.      To  the  clear 
fluid  remaining  add  some  Millon's  reagent  and  heat  cautiously. 
Only  a  slight  red  precipitate  is  formed,  showing  that  the  proteins 
of  saliva  consist  almost  entirely  of  mucin. 

B.  Ptyalin. 

Ptyalin,  or  salivary  amylase,  is  an  enzyme  that  acts 
on  boiled  starch  and  certain  other  polysaccharides,  the 
chief  end  product  being  the  disaccharide  maltose.  It  is 
possible  that  small  amounts  of  glucose  are  also  formed.  It 
is  claimed  by  certain  workers  that  for  the  complete  hydroly- 
sis of  starch  three  ferments  are  necessary,  viz.,  amylase, 


CH.    VIII.]  PTYALIN.  187 

that  converts  starch  into  dextrins  ;  dextrinase,  that  converts 
dextrins  into  maltose  ;  and  maltase,  that  converts  maltose 
into  glucose. 

In  the  case  of  the  action  of  ptyalin  on  starch  as  con- 
ducted in  vitro,  the  final  product  consists  of  about  80  per 
cent,  of  maltose,  the  remaining  20  per  cent,  being  a  com- 
paratively simple  dextrin  called  "  stable  "  dextrin,  owing 
to  its  resistance  to  the  further  action  of  the  enzyme.  But 
if  this  dextrin  be  isolated  the  action  of  ptyalin  is  to  hy- 
drolyse  it  very  slowly  and  incompletely  to  equal  molecular 
parts  of  maltose  and  glucose. 

The  optimum  reaction  for  ptyalin  is  at  PH  =  6*7.* 
The  enzyme  is  rapidly  destroyed  should  the  reaction  be 
markedly  acid  to  this,  as  it  is  during  full  digestion  in  the 
stomach.  But  the  presence  of  proteins,  which  function  as 
buffers,  prevent  the  hydrochloric  acid  secreted  in  the  early 
stages  of  digestion  from  causing  too  high  a  concentration 
of  hydrogen-ions  for  the  action  of  the  ptyalin.  This, 
combined  with  the  absence  of  active  mechanical  move- 
ments in  the  cardiac  end  of  the  stomach,  allows  salivary 
digestion  to  be  carried  on  in  the  stomach  for  about  30 
minutes  after  the  ingestion  of  a  mixed  meal. 

Ptyalin  is  remarkable  in  that  it  is  inactive  in  the 
absence  of  electrolytes.  As  the  author  first  demonstrated, t 
the  influence  of  electrolytes  on  amylolytic  action  is  depen- 
dent on  the  negative  ion  (kation).  These  have  not  all  the 
same  activating  power,  the  chloridion  being  the  most 
effective.  A  satisfactory  explanation  of  this  effect  has 
not  yet  been  advanced. 

The  optimum  concentration  of  sodium  chloride  is 
between  0*02  per  cent,  and  2  per  cent.-,  between  which 
limits  very  slight  differences  can  be  observed,  but  the 
difference  between  the  action  of  the  enzyme  in  a  salt-free 
mixture  and  in  one  containing  0*02  per  cent,  of  sodium 

*  It  has  recently  been  stated  that  the  optimum  reaction  for  malt  diastase 
is  at  PH  =  4-9. 

f  Journ.  of  Physiology,  1903. 


188  COMPOSITION   OF  THE   DIGESTIVE    JUICES.         [cH.    VIII. 

chloride  is  enormous.  It  is  therefore  of  the  utmost 
importance  in  all  quantitative  experiments  on  the  action 
of  the  enzyme  to  ensure  that  about  o'i  per  cent,  of  the  salt 
is  present.  The  beneficial  effect  of  sodium  chloride  on 
salivary  digestion  offers  a  simple  teleological  explanation 
of  the  desire  for  salt  when  eating  carbohydrate  foods. 

The  estimation  of  ptyalin  has  been  attempted  by  a 
variety  of  methods,  none  of  which  are  very  satisfactory. 
The  most  important  of  these  methods  are  : 

1.  Roberts'  A  chromic  Point  method.      A  given  amount 
of  enzyme  is  added  to  a  measured  amount  of  starch  paste 
at  40°  C.     Portions  of  the  digest  are  treated  with  dilute 
iodine  at  intervals  and  the  time  when  the  iodine  fails  to  give 
a  colour  is  noted. 

2.  Wohlgemuth's  method.     A  fixed  amount  of  soluble 
starch  is  digested  with  varying  amounts  of  the  enzyme  for 
a  stated  time.     Iodine  is  added  to  each,  and  the  amount 
of  enzyme  that  just  converts  the  whole  of  the  starch  to 
dextrin  is  determined. 

3.  Reduction  methods.     Starch   is   digested  with  the 
enzyme  and  the  amount  of  maltose  formed  in  a  given  time 
is  determined. 

C.  Lovatt  Evans  (Journal  of  Physiology,  xliv.,  p.  220) 
has  criticised  the  various  methods,  and  claims  that  the 
only  correct  method  is  a  reduction  method  carried  out 
under  certain  defined  conditions  of  starch  concentration, 
temperature,  etc.  His  main  contention  is  that  the  amount 
of  maltose  formed  is  only  proportional  to  the  amount  of 
enzyme  added,  provided  that 

1 .  Less  than  30  per  cent,  of  the  starch  has  been 
converted,    at   which   stage   a   blue  reaction   is   still 
obtained  with  iodine. 

2.  The    digestion    period    is    a    short    one    (ten 
minutes). 

3.  The  concentration  of  the  starch  is  about  3 
per  cent. 


CH.   VITI.]  PTYALIN.  189 

His  objection  to  the  Achromic  Point  method  is  that 
the  end  point  is  very  difficult  to  determine,  especially  with 
weak  enzymes,  and  that  the  digestion  ("  chromic  ") 
period  bears  no  simple  relationship  to  the  concentration 
of  the  enzyme.  This  is  undoubtedly  well-founded,  for 
if  the  enzyme  be  halved  the  chromic  period  is  more  than 
doubled. 

His  objection  to  Wohlgemuth's  method  is  mainly  based 
on  the  fact  that  in  certain  experiments  the  amounts  of 
enzyme  added  are  in  a  geometrical  series,  and  so  the  only 
results  obtainable  will  also  be  in  a  geometrical  progression. 
But  this  is  a  poor  argument,  for  the  experimenter  can  vary 
the  amount  of  enzyme  added  as  he  pleases. 

It  is  curious  that  neither  Wohlgemuth  nor  Lovatt 
Evans  take  any  serious  precautions  to  maintain  the 
optimum  PH  or  salt  content.  In  fact,  certain  results  of 
the  latter  with  the  Achromic  method  may  be  due  in  part 
to  the  dilution  of  the  chlorides  of  the  saliva  and  not  merely 
to  the  dilution  of  the  enzyme. 

After  a  considerable  amount  of  investigation  the 
author  has  decided  to  adopt  a  colorimetric  method  for 
ordinary  work  (see  Ex.  242).  It  is  by  no  means  perfect, 
but  it  is  fairly  rapid,  and  the  end  point  is  more  easily 
determined  than  in  the  Achromic  or  Wohlgemuth's 
method.  The  relationship  between  amount  of  enzyme 
and  rate  of  digestion  is  fairly  exact  for  moderate  changes 
in  dilution. 

234.  Obtain  diluted  saliva  as  follows :  Warm  some  distilled 
water  in  a  beaker  to  about  40°  C.  With  a  portion  of  this  thoroughly 
rinse  out  the  mouth.  Now  take  about  20  cc.  of  the  warm  water 
into  the  mouth  and  move  it  about  by  the  tongue  for  at  least  a 
minute.  Collect  the  fluid  thus  obtained  in  a  clean  beaker,  and 
repeat  the  process  once  or  twice,  depending  on  the  amount  required. 
Transfer  the  whole  to  a  flask,  close  with  the  thumb,  shake  vigorously, 
and  filter. 


COMPOSITION    OF  THE   DIGESTIVE   JUICES.          [CH.  VIII. 

235.  In  a  clean  test-tube  place  5  cc.  of  i  per  cent,  starch  paste, 
freshly  prepared  with  distilled  water  (see  Ex.  135)  and  5  cc.  of  the 
diluted  saliva.     Mix  well,  place  a  glass  tube  or  pipette  in  the  tube, 
and  place  the  test-tube  in  a  water  bath  maintained  at  about  40°  C. 
Place  a  series  of  drops  of  iodine  solution  (about  0-02  N.)  on  a  clean, 
dry,  white  porcelain  or  opal  glass  plate.     From  time  to  time  allow 
a  drop  of  the  digesting  mixture  to  fall  on  to  one  of  the  drops  of 
iodine,  taking  care  that  the  iodine  is  not  transferred  to  the  digesting 
mixture.     A  blue  colour  should  be  produced  at  first,  but  with  the 
drops  added  later  the  colour  becomes  blue-violet,  red-violet,  red- 
brown,  light  brown,  and  finally  no  increase  of  colour  is  obtained. 
If  no  increase  of  colour  is  obtained  with  the  first  drop,  repeat  the 
experiment,  making  the  first  test  immediately  after  the  starch  and 
saliva  have  been  mixed.     If  the  blue  colour  persists  for  a  long  time, 
try  the  effect  of  adding  a  couple  of  drops  of  5  per  cent,  sodium 
chloride  to  the  digesting  mixture. 

When  a  drop  of  the  mixture  fails  to  give  a  colour  with  the 
iodine,  boil  a  few  cc.  with  a  little  Fehling's  solution.  A  well- 
marked  reduction  is  obtained,  showing  that  trie  enzyme  ptyalin  has 
converted  the  starch  into  a  reducing  sugar,  which  is,  however,  not 
glucose,  but  maltose. 

236.  Perform  a  control  test  by  first  boiling  and  then  cooling 
the  saliva  before  adding  it  to  the  starch.     The  colour  with  iodine  is 
always  blue,  and  the  solution  does  not  reduce  Fehling's. 

237.  Achromic  Point   method.       Measure   5   cc.   of    i    per 
cent,  soluble  starch  (see  p.  391)  into  a  test-tube.   Add  2  cc.  of  a  buffer 
solution  of  PH  =  6-7  and  2  cc.  of  0-5  per  cent,  sodium  chloride. 
Place  the  tube  in  a  water  bath  maintained  at  38°  to  40°  C.  for  a  few 
minutes.     Have  ready  a  series  of  test-tubes,  each  containing  about 
3  cc.  of  distilled  water.     To  each  tube  add  a  couple  of  drops  of  dilute 
iodine  (about  o-oi  N.).     To  the  tube  containing  the  warmed  starch 
add  2  cc.  of  the  diluted  saliva,  mix  well,  and  note  the  time.     At 
intervals  transfer  a  drop  or  two  of  the  digesting  mixture  to  one  of 
the  tubes  containing  the  diluted  iodine  by  means  of  a  quill  tube,  and 
shake.     The  colour  obtained  is  blue  at  first.     Determine  the  time 
when  the  addition  ceases  to  produce  any  colour.     This  point,  which 
is  the  moment  when  the  last  trace  of  erythro-dextrin  is  converted 


CH.   VIII. 


ACHROMIC   POINT   METHOD. 


to  achroo-dextrin  and  maltose,  is  known  as  the  achromic  point. 
The  time  that  is  taken  to  reach  this  point  ("  chromic  period  ")  is  a 
measure  of  the  activity  of  the  ferment. 

If  the  chromic  period  is 
less  than  4  minutes,  dilute  the 
enzyme  with  one  or  more 
volumes  of  distilled  water  and 
repeat  the  experiment.  A' 

NOTES. — i.  The  buffer  solu- 
tion is  added  to  maintain  the 
optimum  concentration  of  hydro- 
gen ions.  It  is  prepared  by  treating 
50  cc.  of  0-2  M.  acid  potassium 
phosphate  with  21  cc.  of  0-2  N. 
soda  and  adding  distilled  water  to 
make  a  total  volume  of  200  cc. 
(See  p.  27.) 

2.  The    reason    for    the    ad- 
dition   of   the    sodium    chloride   is 
explained  in  the  text  on  p.  188. 

3.  Starch  paste  can  be  used 
instead  of  soluble  starch,  but  it  is 
difficult  to  measure  accurately  by 
means  of  a  pipette,   owing  to  its 
viscidity. 

4.  The    use    of   the   author's 
"  Distributor  "    (fig.  29)   for   auto- 
matically delivering  a  given  volume 
of  fluid  is  convenient  for  measuring 
about  3  cc.  of  water  into  each  tube. 
Distilled  water  must  be  used,  owing 
to    the    action    of    tap    water    on 
iodine.        The    iodine    should    not 
be    added    until    just    before    the 
addition  of  the  digesting  mixture. 

238.      Repeat  the  above 
experiment,  substituting  2  cc. 
of  distilled  water  for  the  2  cc. 
period  is  much  prolonged. 


Fig.  29.  Cole's  "Distributor"  for  the 
automatic  delivery  of  a  given  vol- 
ume of  fluid. 

The  fluid  is  placed  in  the  beaker 
and  the  air  is  driven  out  of  the  apparatus 
by  repeatedly  compressing  the  rubber 
ball.  On  firmly  pressing  down  the 
hinged  board  a  given  volume  of  fluid 
is  driven  out  of  the  apparatus.  The 
volume  delivered  can  be  varied  by  ad- 
justing the  screw  C.  The  rubber  tubing 
should  be  thick  pressure  tubing  and  as 
short  as  possible.  The  wooden  base 
should  be  screwed  down. 


of  sodium  chloride.     The  chromic 


239.  Determine  the  effect  of  dilution  of  the  enzyme  on  the 
chromic  period.  It  will  be  found  that  the  chromic  period  lengthens 
out  unduly  as  the  ferment  is  diluted ;  that  is,  the  period  is  more 
than  trebled  by  a  three- times  dilution. 


IQ2  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

240.  Determine  the  effect  of  change  of  reaction  on  the  chromic 
period.     It  will  be  found  that  it  is  least  at  PH=  6-7,  lengthening 
out  as  the  solution  is  made  acid  or  alkaline  to  this.     With  small 
changes  of  reaction,  however,  the  activity  is  but  slightly  affected. 
Determine  the  chromic  period  at  PH  =  5-7  and  7-7. 

241.  Wohlgemuth's  method.     Number  a  series  of  clean  dry 
test-tubes  i  to  10. 

Into  i  and  2  measure  I  cc.  of  the  diluted  enzyme. 

Into  2  to  10  measure  i  cc.  of  distilled  water. 

Mix  the  contents  of  2  and  transfer  i  cc.  from  2  to  3. 

Mix  the  contents  of  3  and  transfer  i  cc.  from  3  to  4. 

Continue  in  this  way,  rejecting  i  cc.  of  the  fluid  from  10. 

To  each  tube  add  5  cc.  of  i  per  cent,  soluble  starch  (see  Note  2), 
commencing  with  tube  10.  Mix  the  contents  of  the  tubes  and 
place  them  in  a  water  bath  at  40°  C.,  noting  the  time  when  they  are 
placed  in  the  bath.  Allow  the  tubes  to  remain  in  the  bath  for 
exactly  30  mins.  Remove  the  tubes  and  immerse  them  all  in  cold 
water  to  stop  the  action. 

Arrange  them  in  order  in  a  rack. 

To  each  tube  add  cold  distilled  water  to  about  2  finger-breadths 
from  the  top  of  the  tube.  Now  add  3  drops  of  0-02  N.  iodine  to 
each  tube  and  mix,  commencing  with  the  tube  i.  This  will  probably 
give  no  colour,  whilst  tube  10  will  probably  give  a  deep  blue.  Be- 
tween these  limits  tubes  will  probably  be  found  which  are  red  and 
purple.  Select  the  tube  with  the  lowest  number  that  has  a  blue 
tinge  mixed  with  the  red.  In  the  tube  numbered  one  below  this 
there  has  been  enough  enzyme  to  completely  convert  5  cc.  of  soluble 
starch  to  erythrodextrin  in  30  minutes. 

Wohlgemuth  indicates  the  concentration  of  enzyme  in  the 
following  way.  He  determines  the  volume  of  i  per  cent,  starch  that 
would  be  converted  to  erythrodextrin  by  i  cc.  of  the  enzyme 
solution  in  30  minutes.  He  calls  this  the  "  diastatic  power  "  of  the 
solution,  and  indicates  it  by  D  for  i  per  cent,  and  by  d  for  o-i  per 
cent,  starch.  He  also  indicates  the  temperature  and  period  of 
digestion.  Thus  in  the  above  experiment,  if  the  6th  tube  were 
blue-violet  and  the  5th  tube  were  red,  then  since  the  5th  tube  con- 
tains iV  cc.  of  the  enzyme,  and  this  amount  has  converted  5  cc. 


CH.  viii.]  WOHLGEMUTH'S  METHOD.  193 

of  i  per  cent,  soluble  starch  to  erythro-dextrin  in  30  minutes,  then 
i  cc.  would  convert  16  x  5  =  80  cc.  of  starch.      If  the  temperature 

of  the  bath  is  40°  C,  then  D  ^  =  80. 

30 

NOTES. — i.  In  the  experiment  as  conducted  above,  the  possible  error 
is  nearly  100  per  cent.  A  nearer  approximation  can  be  made  by  repeating  the 
experiment  with  more  gradual  dilutions  that  will  depend  on  the  result  obtained. 
Thus,  if  T\  cc.  gives  a  red  and  ^  cc.  gives  a  violet,  D  may  be  anything  between 
So  and  160.  To  i  cc.  of  the  enyzme  add  15  cc.  of  distilled  water,  mix  well,  and 
measure  i,  0-9,  0-8,  0-7,  0-6  and  0-5  cc.  into  a  series  of  6  tubes.  Make  up 
the  volume  to  i  cc.  in  each  case  by  the  addition  of  distilled  water.  Add  5  cc. 
of  the  soluble  starch  and  repeat  the  experiment.  One  of  the  following  values 
for  D  will  then  be  obtained  :  80,  88,  100,  114,  133,  or  160. 

2.  Though  in  Wohlgemuth's  original  method  the  instructions  are  to 
use  pure  i  per  cent,  soluble  starch,  the  author  finds  that  the  results  obtained 
are  much  more  reliable  if  the  hydrogen-ion  concentration  and  the  salt  content 
be  maintained  at  the  optimum.     The  starch  solution  is  prepared  by  treating 
100  cc.  of  2  per  cent,  soluble  starch  (see  p.  391)  with  25  cc.  of  a  buffer  solution 
of  PH  =  6-7  (see  Note  i,  Ex.  237),  25  cc.  of  i  per  cent,  sodium  chloride  and 
50  cc.  of  distilled  water.     The  starch  solution  should  be  freshly  prepared. 

3.  It  is  important  to  add  exactly  the  same  amount  of  iodine  to  each 
tube  in  all  the  experiments.     The  iodine  should  be  measured  by  means  of  a 
dropping  pipette  (see  fig.  5). 

242.  The  Method  of  "  the  first  change."  Measure  10  cc.  of  2 
per  cent,  soluble  starch  (see  p.  391)  into  a  test-tube.  Add  2  cc.  of 
a  buffer  solution  of  PH  =  6-7  (seeNote  i,  Ex.  237)  and  2  cc.  of  0-5  per 
cent,  sodium  chloride.  Place  the  tube  in  a  water  bath  at  40°  C.  for 
some  minutes  until  it  has  attained  the  bath  temperature.  Have 
ready  a  series  of  tubes  containing  3  cc.  of  distilled  water,  to  each  of 
which  has  been  added  a  single-  drop  of  o-oi  N.  iodine  by  means  of  a 
dropping  pipette  (fig.  5).  Now  add  2  cc.  of  the  enzyme  to  the 
starch  tube  by  means  of  a  2  cc.  pipette,  discharging  this  by  blowing 
through  it  for  a  second  into  the  solution.  Note  the  time  of  the 
addition  of  the  enzyme.  Seal  the  tube  by  the  thumb  and  mix  by 
shaking  ;  immediately  replace  the  tube  in  the  water  bath.  Insert  a 
quill  tube.  At  the  end  of  i  minute  allow  a  single  drop  of  iodine  to 
fall  into  one  of  the  iodine  tubes,  shake  by  mixing,  and  place  this  tube 
in  the  first  hole  of  a  test-tube  rack.  At  the  end  of  2  minutes,  allow 
another  drop  to  fall  into  another  iodine  tube,  shake  and  place  this 
in  the  second  hole  of  the  rack.  Continue  in  this  way  until  the 
colour  given  by  the  digestion  mixture  is  violet.  Now  examine  the 
tubes  carefully  in  diffuse  daylight,  and  select  the  tube  in  which  the 


194  COMPOSITION   OF  THE  DIGESTIVE  JUICES.  [CH.  VIII. 

pure  "starch-blue"  colour  has  just  definitely  changed  and  a  very 
slight  tinge  of  violet  is  perceptible.  From  the  position  of  this  tube 
in  the  rack  the  time  required  to  produce  this  effect  is  obtained.  It 
is  a  measure  of  the  activity  of  the  enzyme. 

If  the  digestion  time  is  much  less  than  10  minutes,  repeat  the 
experiment  with  a  diluted  enzyme,  attempting  to  arrive  at  a  diges- 
tion period  of  about  10  minutes.  Thus,  if  in  the  first  experiment 
the  third  tube  shows  the  change,  dilute  3  cc.  of  the  enzyme  with 
7  cc.  of  distilled  water.  If  the  fourth  tube  shows  the  change,  dilute 
4  cc.  with  6  cc.  of  distilled  water,  and  so  on.  In  the  second  experi- 
ment it  is  advisable  to  take  a  sample  drop  every  half-minute  when 
near  the  expected  end  point.  These  "half-minute  "  tubes  can  be 
subsequently  identified  by  placing  them  behind  the  "  minute " 
tubes  in  a  double  test-tube  rack. 

NOTES. — i.  The  drops  of  iodine  should  not  be  added  to  the  distilled 
water  in  the  tubes  until  just  before  (or  even  after)  the  enzyme  has  been 
added  to  the  starch  tube.  It  is  important  to  add  only  one  drop  to  each 
tube. 

2.  The  author's  "  Distributor  "  (fig.  29)  is  convenient  for  measuring 
the  3  cc.  of  distilled  water. 

3.  The  effect  of  the  concentration  of  hydrogen-ions  can  be  investigated 
by  varying  the  PH  of  the  buffer  solution  added  (see  Ex.  240). 

4.  The  author  suggests  that  the  unit  of  amylase  should  be  taken  as  the 
amount  which  can  convert  TO  cc.  of  2  per  cent,  soluble  starch  to  the  stage 
obtained  in  10  minutes.     So  if  2  cc.  of  a  4  in  10  dilution  effects  the  change  in 

10        10 
9-5  mins.,  then  2  cc.  contains    -  x  —  =  2-62  units.     This  can  be  expressed, 

A  =  131  per  100  cc. 

C,     Gastric  Juice. 

Human  gastric  juice  has  been  obtained  in  certain  cases 
in  which  an  artificial  opening  into  the  stomach  has  been 
necessitated  owing  to  stricture  of  the  oesophagus.  It  con- 
tains about  0-37  per  cent,  of  hydrochloric  acid,  small 
amounts  of  the  chlorides  of  sodium,  calcium  and  potassium, 
traces  of  phosphates  and  of  mucin,  together  with  the 
enzymes,  pepsin,  rennin  and  lipase. 

From  a  practical  standpoint  the  composition  of  the 
gastric  contents  at  stated  times  after  the  ingestion  of 


CH.  VIII.]  GASTRIC   JUICE.  IQ5 

standard  test  meals  is  more  important  than  that  of  the 
juice  as  actually  secreted.  The  hydrochloric  acid,  secreted 
mainly  by  the  oxyntic  cells  of  the  fundus,  neutralises  the 
alkaline  salts  of  the  saliva  to  form  sodium  chloride.  It 
also  combines  with  the  proteins  of  the  saliva  and  of  the 
food  to  form  acid-protein  compounds.  These  function  as 
weak  acids.  In  this  way  the  percentage  amount  of  free, 
uncombined  hydrochloric  acid  in  the  gastric  contents  may 
be  very  considerably  less  than  in  the  naturally  secreted 
juice.  Further  alterations  are  brought  about  by  the 
fermentation  of  the  carbohydrates  to  lactic  acid.  This  is 
mainly  caused  by  certain  organisms  called  sarcinae,  which 
are  very  commonly  present  in  the  stomach.  If  the  starchy 
foods  are  thoroughly  masticated  and  mixed  with  the 
saliva,  the  polysaccharides  are  rapidly  broken  down  by 
the  ptyalin  to  maltose.  This  is  passed  through  the  pylorus 
and  leaves  little  for  the  sarcinae  to  ferment.  The  presence 
of  much  lactic  acid  generally  indicates  a  diminished  secre- 
tion of  hydrochloric  acid,  which  inhibits  the  action  of  the 
lower  organisms. 

Ewald  test  meal.  The  patient  under  investigation  is 
starved  for  at  least  12  hours.  The  meal  consists  of  400  cc. 
of  weak  tea,  without  milk  or  sugar,  and  50  grams,  of  dry 
toast,  which  should  be  well  masticated.  After  i  hour  the 
gastric  contents  are  removed  by  syphonage  through  a  soft 
rubber  stomach  tube. 

The  fluid  obtained  is  measured  and  filtered.  The 
normal  physiological  volume  is  between  40  and  70  cc.  A 
marked  increase  in  volume  probably  indicates  motor 
insufficiency  of  the  stomach,  or  hypersecretion,  the  latter 
generally  being  associated  with  an  increased  amount  of 
hydrochloric  acid  (hyperchloridia). 

Chemical  examination.  The  reaction  of  the  filtered 
juice  is  nearly  always  acid  to  litmus  paper.  The  percent- 
age amount  of  the  following  substances  must  be  deter- 
mined : 


196  COMPOSITION    OF   THE    DIGESTIVE   JUICES.  [CH.  VIII. 

A.  Total  acidity.     (Ex.  243.) 

B.  Total  chlorides.     (Ex.  244.) 

C.  Mineral  chlorides.     (Ex.  245.) 

D.  Active  hydrochloric  acid.     (B  —  C.) 

E.  Free  hydrochloric  acid.     (Ex.  246.) 

F.  Combined  hydrochloric  acid.     (D  —  E.) 

G.  Abnormal  acidity.     (A  -  D.) 

It  is  usual  to  express  all  these  results  in  terms  of  grams, 
of  hydrochloric  acid  per  cent. 

Total  acidity  is  determined  by  titration  with  0*1  N. 
soda,  the  indicator  being  phenol-phthalein. 

Total  chlorides  is  obtained  by  adding  i  cc.  of  a  saturated 
solution  of  sodium  carbonate  to  100  cc.  of  the  nitrate, 
evaporating  on  the  water  bath,  heating  to  a  dull  red  heat 
and  estimating  the  total  chlorides  by  Volhard's  method. 

Mineral  chlorides  are  determined  in  a  similar  way, 
except  that  the  sodium  carbonate  is  not  added.  Most 
of  the  free  hydrochloric  acid  is  evolved  on  heating  to 
dryness.  On  incineration  the  protein  matter  is  destroyed 
and  any  hydrochloric  acid  combined  with  it  is  evolved. 
The  only  chloride  left  is  that  which  was  originalry  present 
in  the  form  of  non-volatile  chlorides  of  sodium,  etc.  The 
difference  between  this  and  the  former  estimation  is  a 
measure  of  free  hydrochloric  acid  plus  that  combined  with 
proteins,  the  sum  of  these  being  known  as  "  active  hydro- 
chloric acid."  Since  active  hydrochloric  titrates  with  soda 
to  phenol  phthalein,  the  difference  between  total  acidity 
and  active  hydrochloric  is  a  measure  of  the  abnormal  acids 
present,  generally  lactic  acid. 

In  a  few  cases  the  author  has  observed  that  the  active  hydrochloric  acid 
is  greater  than  total  acidity.  This  puzzling  result  is  probably  due  to  the 
presence  of  small  amounts  of  ammonium  chloride,  which  is  volatile  at  a  low 
red  heat,  and  would  be  removed  in  the  estimation  of  mineral  chlorides.  After 
treatment  with  sodium  carbonate,  the  ammonium  chloride  would  be  con- 
verted to  sodium  chloride.  In  such  a  case,  the  mineral  chlorides  work  out  too 
low  and  the  active  hydrochloric  proportionally  too  high,  and  in  the  absence  of 
lactic  acid  the  active  hydrochloric  acid  will  exceed  the  total  acidity. 

Free  hydrochloric  acid.  The  only  satisfactory  clinical 
method  for  the  estimation  of  this  is  b}^  use  of  Gunzberg's 


CH.  VIII.]  FREE   HYDROCHLORIC   ACID.  IQ7 

reagent.     This    is    an    alcoholic    solution    of  phloroglucin 
(symmetrical  tri-hydroxy  benzene,  C6H3(OH)3  and  vanillin 

-OCH3 
CH        -OH 


When  evaporated  with  hydrochloric  acid  on  the  water 
bath  these  condense  to  form  a  brilliant  red  compound, 

(-20**  18^8  • 

2C6H603  +  C8H803  =  C20H1808  +  H20. 

In  the  absence  of  free  hydrochloric  acid  only  a  brown 
colour  is  obtained.  A  brown  colour  also  develops  in  the 
presence  of  hydrochloric  acid  if  the  mixture  be  over- 
heated. 

There  are  two  methods  of  applying  the  test.  One  is  to 
dilute  the  solution  until  it  fails  to  give  a  positive  reaction, 
assuming  that  it  is  just  positive  with  0-0004  N.HC1 
(0*00146  per  cent.).  The  other,  and  better,  method  is  to 
titrate  a  measured  sample  with  soda  until  a  drop  of  the 
titrated  mixture  just  fails  to  give  the  reaction. 

From  comparisons  with  the  electrical  method  of 
measuring  hydrogen-ion  concentration  (p.  19)  it  would 
seem  that  the  estimation  by  Gunzberg's  reagent  is  accurate 
when  the  acidity  is  high  or  moderate,  but  that  it  gives  too 
low  a  result  with  gastric  contents  relatively  deficient  in 
hydrochloric  acid. 

At  one  time  it  was  claimed  that  Toepfer's  reagent 
(p.  22)  only  reacted  with  free  hydrochloric  acid.  It  has 
now  been  shown  that  both  this  indicator  and  also  congo  red 
react  with  lactic  acid  if  this  be  sufficiently  concentrated, 
and  that  the  amount  of  free  hydrochloric  acid  as  deter- 
mined by  their  use  may  be  far  in  excess  of  the  amount 
actually  present. 

Results  of  analysis.  In  normal  cases  the  following  may 
be  taken  as  the  limits,  the  results  being  expressed  in  grams, 
of  HC1  per  cent. 


ig8  COMPOSITION   OF  THE  DIGESTIVE  JUICES.  [CH.  VIII. 

Total  acidity    ..  ..  0-14  to  0-26 

Total  chlorides  . .  0-2     to  0-3 

Mineral  chlorides  ..  0-09  to  0-12 

Active  HC1      ..  ..  0-14  to  0-26 

Free  HC1          ..  ..  0-07  to  0-15 

G.  Graham  [Quarterly  Journal  of  Medicine,  IV.,  p.  315 
(1911)  ],  has  published  an  interesting  series  of  cases.  The 
average  results  he  obtained  are  as  follows  : 

Total          Mineral        Active  Ratio 

Chlorides.     Chlorides.         HC1.       Active:  Mineral. 

Gastric  ulcer         . .  0-335  °*°99  0-236  238  :  100 

Dilated  stomach  ..  0-256  0-094  0-182  194:  ooo 
Carcinoma  of 

stomach..  0-197  0-142  0-052  37:  100 

It  will  be  noted  that  in  carcinoma  of  the  stomach  the 
ratio  of  active  HC1  to  mineral  chlorides  is  markedly  sub- 
normal, due  to  a  decreased  secretion  of  HC1  and  the 
neutralisation  of  a  good  deal  of  this  by  some  alkaline 
secretion.  This  change  would  seem  to  be  more  diagnostic 
than  the  absence  of  free  HC1,  since  the  latter  occurs  in 
certain  other  pathological  conditions.  In  gastric  and 
duodenal  ulcer  there  is  an  increase  in  the  amount  of  free 
and  active  HC1,  and  the  active  mineral  ratio  is  apt  to  be 
above  normal. 

Mixture  for  analysis.  The  following  mixture  can  be  analysed  for  the 
purpose  of  acquiring  the  necessary  technique : — 

1  per  cent,  sodium  chloride       . .          . .  14  cc. 
o-i  N.  HC1              50  cc. 

2  per  cent,  peptone         . .          . .          . .  26  cc. 

Distilled  water  to  make             . .          . .  100  cc. 

243.  Total  acidity.  To  10  cc.  add  4  drops  of  a  0-5  per  cent, 
solution  of  phenol  phthalein  in  50  per  cent,  alcohol.  Titrate  with 
o-i  N.  soda  until  a  faint  but  definite  pink  tinge  is  obtained.  The 
end  point  is  not  very  sharp,  owing  to  the  buffer  action  of  the  peptone. 

Calculation.     I  cc.  of  o-i  N.  NaOH  =  0-00365  gram.  HC1. 
Express  the  result  in  grams,  of  HC1  per  100  cc. 


CH.  VIII.]  TOTAL  CHLORIDES.  199 

244.    Total  Chlorides  by'the  Prout- Winter  method, 

Principle  of  Volhard's  process  for  the  estimation  of  chlorides.  A  measured 
amount  of  standard  silver  nitrate  is  added  to  a  known  amount  of  the  solution, 
or  to  a  properly  prepared  extract  of  a  known  amount  of  material.  The 
amount  of  silver  used  must  be  more  than  enough  to  completely  precipitate 
the  whole  of  the  chlorides.  Iron  alum  is  added  (to  serve  as  an  indicator),  the 
mixture  is  made  acid  with  nitric  acid  (to  prevent  the  precipitation  of  other 
substances),  and  the  mixture  made  up  to  a  definite  volume.  The  precipitate 
of  silver  chloride  is  filtered  off  and  the  amount  of  silver  in  an  aliquot  portion 
of  the  filtrate  determined  by  titration  with  standard  thiocyanate  solution. 
From  the  amounts  of  standard  silver  originally  used  and  that  found  in  the 
filtrate  the  amount  precipitated  by  the  chlorides  in  the  material  taken  can  be 
calculated. 

Solutions  required,  o-i  N.  Silver  nitrate.  Dissolve  16-99  grams,  of  pure 
fused  silver  nitrate  in  distilled  water  and  make  the  volume  up  to  i  litre.  The 
solution  should  be  kept  in  the  dark. 

i  cc.  =  0-00365  gram.  HC1. 

o-i  N.  thiocyanate.  Dissolve  about  15  grams,  of  potassium,  or  about 
10  grams,  of  ammonium  thiocyanate,  in  a  litre  of  distilled  water  and 
mix  thoroughly.  Standardise  this  in  the  following  way : — Measure  out 
20  cc.  of  the  standard  silver  nitrate  into  a  150  cc.  beaker  or  Erlenmeyer 
flask.  Add  about  60  cc.  of  distilled  water,  5  cc.  of  pure  nitric  acid, 
and  5  cc.  of  a  cold  saturated  solution  of  iron  alum.  Titrate 
with  the  thiocyanate  from  a  burette.  A  white  precipitate  of  silver 
thiocyanate  is  formed.  Continue  to  add  the  thiocyanate  until  a 
faint  permanent  pink  (due  to  ferric  thiocyanate)  is  obtained.  Let  x  cc. 
be  the  amount  of  thiocyanate  required.  To  i  litre  of  the  solution  add 

—  cc.  of  distilled  water.     The  mixture  should  now  be  o-i  N.     It 

is  advisable  to  check  the  strength  once  more  against  the  standard  silver. 
Iron  alum.     A  cold  saturated  solution  in  distilled  water. 
Pure  nitric  acid,  free  from  chlorides. 

Method,  To  10  cc.  of  the  filtered  fluid  in  a  porcelain  or  silica 
crucible  (not  in  an  evaporating  basin)  add  i  cc.  of  a  saturated  solu- 
tion of  sodium  carbonate.  Evaporate  to  complete  dry  ness  on  a 
boiling  water  bath.  Support  the  crucible  on  a  pipe  clay  triangle, 
and  cautiously  heat  with  a  Bunsen  burner.  Gradually  raise  the 
temperature  until  the  mass  has  completely  carbonised.  The  heating 
should  be  continued  until  as  much  as  possible  of  the  carbon  has 
disappeared  and  the  whole  has  been  raised  to  a  dull  red  heat. 
Remove  the  flame  and  allow  the  crucible  to  cool  until  it  can  be 
handled.  Add  about  10  cc.  of  distilled  water  and  carefully  stir  with 
a  glass  rod.  Pour  the  fluid,  together  with  the  little  pieces  of  carbon, 
into  a  100  cc.  graduated  flask,  using  a  small  funnel.  Repeat  the 
extraction  with  another  10  cc.  of  water :  then  with  5  cc.  of  pure 
nitric  acid  and  5  cc.  of  water :  then  twice  more  with  water.  Wash 


200  COMPOSITION    OF   THE   DIGESTIVE    JUICES.  [CH.  VIII. 

down  the  funnel  with  a  little  water  and  remove  it.  To  the  flask 
add  20  cc.  of  the  standard  silver  nitrate,  measuring  this  by  means 
of  a  pipette.  Then  add  5  cc.  of  the  iron  alum,  and  make  the  volume 
up  to  the  mark  with  distilled  water.  Mix  thoroughly  and  filter 
through  a  dry  paper  into  a  clean,  dry,  100  cc.  measuring  cylinder. 
Collect  at  least  90  cc.  of  the  fluid  and  note  its  exact  volume.  Trans- 
fer it  to  a  beaker  or  flask,  wash  out  the  cylinder  with  a  little  water 
and  titrate  with  the  thiocyanate  from  a  burette  until  a  faint  but 
permanent  pink  colour  is  obtained. 

Calculation.    Suppose  that  92  cc.  of  the  filtrate  were  taken, 
and  that  this  required  11*4  cc.  of  thiocyanate. 

100 

Then  100  cc.  of  the  filtrate  would  require  11-4  x =12-4  cc. 

92 

So  the  chlorides  have  precipitated  20  -  12-4  =  7-6  cc.  of  the 

standard  AgNO3. 

So  total  chlorides  in  10  cc.  =  7-6  x  0-00365  gram.  HC1. 
So  total  chlorides  in  100  cc.  =  0-277  gram.  HC1. 

245.  Mineral  Chlorides.     Repeat  the  above  exercise  in  every 
particular,  except  that  the  sodium  carbonate  is  not  added.     The 
calculation  is  made  in  the  same  way  as  in  the  above  case. 

Active   HC1   is  the  difference  between  these  two  results.     It 
should  be  compared  with  the  result  of  Ex.  243. 

246.  Free  Hydrochloric  acid.     To  10  cc.  of  the  filtered  fluid 
in  a  small  beaker  add  i  cc.  of  o-i  N.  sodium  hydroxide  from  a 
burette.     Stir  well  and  place  a  drop  of  the  mixture,  together  with  a 
drop  of  freshly  prepared  Gunzberg's  reagent  (see  p.  390) ,  into  a  white 
evaporating  basin.     Heat  on  a  boiling  water  bath.     If  free  HC1  is 
still  present  the  film  will  develop  a  brilliant  carmine  tinge  as  it  dries. 
In  that  case  add  another  cc.  of  the  NaOH,  and  repeat  the  evaporation 
with  another  drop  of  the  reagent.     Proceed  in  this  way,  adding  i  cc. 
of  soda  at  a  time  until  a  negative  test  is  obtained.    Then  add  0-5 
or  less  of  o-i  N.  HC1  from  a  burette  and  repeat  the  test.     If  it  is 
now  positive  add  o-i  or  0-2  cc.  of  soda  and  repeat  again.     It  is 
necessary  to  determine  the  amount  of  soda  and  HC1  that  must  be 


CH.  VIII.]  PEPSIN.  2OI 

added  so  that  the  test  is  just  negative.  With  a  little  experience 
one  can  judge  how  near  one  is  to  the  end  point,  from  the  rapidity 
with  which  the  red  colour  develops  and  from  the  intensity  obtained. 

Calculation,  x  cc.  of  soda,  together  with  y  cc.  of  HC1,  just  fail 
to  give  a  positive  reaction. 

(x  —  y)  cc.  o-i  N.  soda  are  required  to  neutralise  the  free  HC1 
in  10  cc.  of  the  fluid. 

Free  HC1  =  (x  —  y)  x  0-0365  gram,  per  cent. 

D,    Pepsin, 

Pepsin  is  the  proteolytic  enzyme  secreted  by  the  chief 
or  peptic  cells  of  the  gastric  glands.  These  cells  elaborate 
the  zymogen,  pepsinogen,  which  is  converted  to  pepsin  by 
hydrochloric  acid. 

Pepsin  is  remarkable  in  only  acting  in  a  decidedly 
acid  medium,  the  optimum  PH  being  about  i  -4.  Not  only 
is  it  inactive  in  neutral  or  alkaline  solutions,  but  the  latter 
rapidly  destroy  it.  Pepsinogen  is  much  more  stable  to 
alkaline  solutions,  and  can  thus  be  distinguished  from 
pepsin.  Acids  other  than  hydrochloric  can  be  employed 
in  artificial  digestion  experiments,  but  not  so  successfully. 
With  such  weak  acids  as  acetic  and  butyric  the  digestive 
action,  even  in  4  per  cent,  of  the  acid,  is  very  feeble,  since 
the  requisite  hydrogen-ion  concentration  cannot  be  ob- 
tained. Neutral  salts  inhibit  the  action  of  pepsin,  this 
being  in  marked  contrast  to  the  influence  of  sodium  chloride 
on  ptyalin. 

The  chemical  nature  of  pepsin  has  not  yet  been  estab- 
lished, it  being  almost  impossible  to  separate  it  from 
substances  on  to  which  it  is  adsorbed.  It  may  eventually 
transpire  that  the  fact  that  all  preparations  contain  iron 
and  chlorine  may  not  be  without  significance. 

Commercial  pepsin  is  prepared  by  extracting  the 
mucous  membrane  of  hogs'  stomachs  with  dilute  hydro- 
chloric acid,  filtering,  and  concentrating  under  reduced 
pressure  at  low  temperatures,  a  large  surface  for  evapora- 
tion being  provided.  A  more  active  product  can  be 


2O2  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

obtained  by  extracting  the  washed  and  minced  mucosa 
with  3  parts  by  weight  of  5  per  cent,  alcohol  for  4  hours, 
filtering  and  concentrating  under  reduced  pressure. 

The  products  of  action  of  pepsin  differ  with  the  nature 
of  the  protein  undergoing  digestion.  The  distinguishing 
feature  of  peptic  digestion  is  that  the  final  products  consist 
mainly  of  simple  proteins  called  peptones  and  polypep tides. 
It  is  true  that  in  artificial  digestions  over  long  periods, 
traces  of  amino-acids  are  produced.  But  this  action  is  of 
little  physiological  importance  compared  to  the  correspond- 
ing action  of  trypsin  and  erepsin.  An  account  of  some  of 
the  products  formed  during  the  peptic  digestion  of  fibrin  is 
given  on  p.  53. 

It  is  possible  that  pepsin  does  not  break  the  ordinary 
pep  tide  linkage  (see  Note  5,  Ex.  24).  It  is  significant  that 
it  has  no  action  on  any  artificial  polypeptide,  many  of 
which  are  split  into  their  constituent  amino-acids  by 
trypsin  or  erepsin.  The  proteoses  and  peptones  formed 
by  peptic  digestion  may  be  united  in  the  intact  protein 
molecule  by  some  special  form  of  linkage  which  is  readily 
attacked  by  pepsin.  This  has  not  yet  been  satisfactorily 
demonstrated,  but  from  various  considerations  it  would 
seem  to  be  highly  probable. 

Pepsin  can  be  estimated  by  a  variety  of  methods.  A 
very  well-known  method  is  that  of  Mett.  Egg-white  is 
drawn  up  into  fine  glass  tubes  and  coagulated  by  heat. 
Lengths  are  cut  off,  immersed  in  the  solution  for  a  stated 
time,  and  the  amount  of  egg-white  digested  determined 
by  measurement.  Experience  has  shown  that  the  length 
of  egg-white  digested  varies  as  the  square  root  of  the 
amount  of  pepsin  present,  this  relationship  being  known 
as  the  "  Schutz-Borissow  law."  The  preparation  of 
satisfactory  tubes  is  not  a  simple  matter.  J.  Christiansen 
(Biochem.  Zeitschrift,  XLVL,  p.  257)  has  described  a 
method  of  standardising  the  egg  tubes,  but,  perhaps  in 
spite  of  this,  the  method  seems  to  be  falling  into  disuse. 

The  edestin  method  of  Fuld  (Ex.  250)  is  reliable,  except 


CH.  VIII.]  PEPSIN.  203 

for  solutions  containing  small  amounts  of  pepsin  and 
relatively  large  amounts  of  salts  and  other  substances. 
These  partially  precipitate  the  edestin  and  render  it 
impossible  to  make  satisfactory  observations. 

For  clinical  purposes  the  author  suggests  that  the 
simplest  method  of  estimating  pepsin  would  be  to  determine 
the  time  required  for  the  clotting  of  "  calcified  "  milk.  It 
is  true  that  the  method  would  not  distinguish  pepsin  from 
rennin,  but  it  is  improbable  that  rennin  is  secreted  by  the 

adult  human  stomach  (see  p.  207). 

/ 

For  the  following  experiments  use  a  0-5  per  cent, 
solution  of  commercial  pepsin  (Armour's)  in  water. 

247.  Place  equal  amounts  of  fresh  washed  fibrin  in  four  test- 
tubes  labelled  A,  B,  C,  and  D. 

To  A  add  5  cc.  of  pepsin  and  5  cc.  of  0-4  per  cent.  HC1. 

To  B  add  5  cc.  of  pepsin  and  5  cc.  of  water. 

To  C  add  5  cc.  of  water  and  5  cc.  of  0-4  per  cent.  HC1. 

To  D  add  5  cc.  of  pepsin  that  has  been  boiled  and  then  cooled, 
and  5  cc.  of  0-4  per  cent.  HC1. 

Place  the  four  tubes  in  a  water  bath  at  40°  C.  for  at  least  thirty 
minutes. 

Note  that  in 

A,  the  fibrin  swells  up,  becomes  transparent,  and  then  dissolves ; 

B,  the  fibrin  is  unaltered; 

C,  the  fibrin  swells  up,  becomes  transparent,  but  does  not  dis- 
solve ; 

D,  the  fibrin  is  like  that  in  C. 

NOTE. — These  exercises  show  that  neither  0-2  per  cent.  HC1  alone,  nor 
pepsin  alone,  can  digest  fibrin,  but  that  pepsin  in  the  presence  of  0-2  per  cent. 
HC1  has  this  property.  In  D  the  ferment  pepsin  has  been  destroyed  by  boiling. 
Fibrin  is  obtained  by  whipping  blood  at  a  slaughter  house  with  a  bundle  of 
twigs,  feathers,  etc.  The  blood  must  be  whipped  as  soon  as  it  is  shed.  The 
mass  of  fibrin  is  placed  on  a  sieve  and  thoroughly  washed  in  running  water  to 
remove  the  haemoglobin.  It  is  then  chopped  up  on  a  board  into  small 
pieces. 


204  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

248.  The  detection  of  pepsin,     Obtain  some  fibrin  that  has 
been  stained  with  carmine  (see  Note  below).     Treat  the  ferment 
solution  with  the  same  volume  of  0-4  per  cent.  HC1.     Divide  this 
into  two  equal  portions  and  label  them  A  and  B.     Boil  B  for  a 
minute,  and  cool  the  tube.     To  each  tube  add  a  few  flakes  of  the 
stained  fibrin.     Place  them  on  the  warm  bath  for  ten  minutes. 
Shake  and  observe  the  colour  of  the  fluid.     In  A  it  will  be  red.     In 
B  it  will  be  almost  or  quite  colourless. 

NOTE. — The  carmine  solution  for  staining  fibrin  is  prepared  by  dissolving 
i  gram,  of  carmine  in  about  i  cc.  of  ammonia  and  adding  400  cc.  of  water. 
The  solution  is  kept  in  a  loosely-stoppered  bottle  till  the  smell  of  ammonia  has 
become  faint.  Fresh  washed  fibrin  is  chopped  finely,  placed  in  the  carmine 
solution  for  twenty-four  hours,  strained  off  and  washed  in  running  water  till  the 
washings  are  colourless.  If  not  required  immediately,  it  should  be  kept  under 
ether  and  washed  with  water  before  use.  It  cannot  be  used  for  testing  for 
trypsin,  owing  to  the  solubility  of  the  dye  in  alkalies. 

249.  Destruction  of  pepsin  by  alkalies.     To  5  cc.  of  the  pepsin 
solution  add  i  per  cent,  caustic  soda  drop  by  drop  until  the  reaction 
is  just  faintly  alkaline  to  litmus  paper.     Place  in  the  warm  bath 
for  10  minutes.     Make  the  reaction  just  acid  to  litmus  by  the 
addition  of  0-4  per  cent,  hydrochloric  acid,  and  then  add  to  the 
mixture  its  own  volume  of  the  same  acid.     Add  some  carmine  fibrin 
and  place  the  tube  in  the  warm  bath.     The  fibrin  does  not  dissolve 
owing  to  the  destruction  of  the  pepsin  by  the  alkali. 

NOTE. — In  light  of  the  above  experiment,  it  is  unnecessary  to  test  an 
alkaline  solution  for  pepsin. 

250.  The  estimation  o!  Pepsin  by  Fuld's  method. 

Principle.  An  acid  solution  of  edestin  (the  protein 
of  hemp  seeds)  is  precipitated  by  sodium  chloride  :  the 
peptic  digestion  products  are  not  precipitated. 

Solutions  required. 

1.  Hydrochloric  acid.     Dilute  30  cc.  of  N/io  HC1  to  100  cc.  with  distilled 
water. 

2.  o- 1  per  cent,  solution  of  edestin.     Dissolve  o-i  gram,  of  pure  edestin 
in  100  cc.  of  the  hydrochloric  acid  at  boiling  point.     Cool  and  make  up  to 
100  cc.  with  the  hydrochloric  acid.     If  the  solution  is  not  clear  it  must  be 
filtered. 

3.  Saturated  (33  per  cent.)  solution  of  sodium  chloride. 

Methoi.     Number  a  series  of  clean  tubes  from  i  to  10.     Into 


CH.  VIII.]  ESTIMATION   OF   PEPSIN.  2O5 

tubes  2  to  10  measure  I  cc.  of  the  hydrochloric  acid.  Into  tubes  i 
and  2  measure  i  cc.  of  the  gastric  juice.  Mix  the  fluid  in  tube  2  and 
transfer  i  cc.  to  tube  3.  Mix  this  and  transfer  i  cc.  of  the  mixture 
to  tube  4.  Proceed  in  this  way  till  each  tube  contains  i  cc:  of  fluid 
and  each  tube  contains  one-half  of  the  amount  of  enzyme  present 
in  the  tube  with  the  next  lower  number,  i  cc.  of  tube  10  being 
rejected.  To  each  tube  add  2  cc.  of  the  edestin  solution.  Mix  and 
allow  the  tubes  to  stand  at  room  temperature  (15  to  17°  C.)  for  30 
minutes.  To  each  tube  add  10  drops  of  the  sodium  chloride.  The 
tubes  with  low  numbers  are  probably  clear,  whilst  the  tubes  with 
high  numbers  are  cloudy.  Note  the  tube  with  the  lowest  number 
that  shows  a  cloud.  The  tube  with  the  number  next  below  it  has  an 
amount  of  gastric  juice  that  just  digests  2  cc.  of  the  edestin  in  30 
minutes.  Thus  the  number  of  cc.  of  edestin  digested  by  i  cc.  of 
gastric  juice  can  be  calculated. 

This  is  best  denoted  by  pe  — . 

30 

Thus  if  tube  6  shows  a  cloud,  then  in  tube  5  (containing  i-i6th 
cc.  gastric  juice)  digestion  is  complete.  Supposing  the  temperature 

16°      2 
is  16°  C.,  then  pe  -  7  =  ... .  =  32. 

30        TS 
251.    The  estimation  of  Pepsin  by  Mett's  method. 

Preparation  of  the  tubes.  The  whites  of  several  new-laid  eggs  are  beaten 
to  break  the  membranes,  strained  through  linen  or  muslin  and  allowed  to 
stand  till  free  from  air  bubbles.  The  liquid  is  then  drawn  up  into  lengths  of 
glass  tubing  with  an  internal  diameter  of  between  i  and  2  mm.  Each  length 
is  laid  flat  on  a  piece  of  wire  gauze,  so  arranged  that  it  can  be  dropped  into  a 
saucepan  of  hot  water,  having  a  double  bottom  ("  porridge  saucepan  ").  The 
water  in  the  saucepan  is  boiled  and  allowed  to  stand  till  that  in  the  inner 
vessel  has  cooled  to  85°  C.  The  gauze  with  the  prepared  tubes  is  then  placed 
in  this  inner  vessel,  and  allowed  to  stand  till  the  water  is  quite  cold.  The 
tubes  can  be  preserved  by  sealing  the  ends  with  shellac. 

Method  of  estimation .  Cut  off  lengths  of  2  cms.,  breaking  the 
tubes  sharply  to  get  an  even  edge  of  coagulated  egg-white. 

Measure  10  to  20  cc.  of  the  ferment  into  a  small  Erlenmeyer 
flask.  In  it  place  two  of  the  tubes  of  egg-white,  shake  and  cork, 
and  place  the  flask  in  a  thermostat  at  40°  C.  for  24  hours.  The 
mixture  must  not  be  shaken  during  the  digestion.  Measure  the 
length  of  the  tube  (T)  and  of  the  remaining  egg-white  (W)  by  means 


2O6  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

of  a  millimetre  scale  and  a  magnifying  glass.  T  —  W  =  the  amount 
of  protein  digested  (D).  Take  the  average  for  the  two  tubes.  D 
varies  as  the  square  root  of  the  amount  of  ferment  present. 

N 
NOTES. — Filtered  gastric  contents  should  be  diluted  with  —  HC1  in  the 

proportion  of  i  cc.  of  gastric  contents  to  15  cc.  of  acid. 

For  practice  use  a  0-5  per  cent,  solution  of  commercial  pepsin  in  —  HC1. 

Dilute  i,  4,  and  9  cc.  to  16  cc.  with  HC1.  The  amounts  of  egg-white 
digested  should  be  as  V?  :  Vj :  Vg  i.e.  as  i :  2  :  3. 

252.    The  estimation  of  Pepsin  by  Calcified  Milk. 

Preparation  of  the  milk.  To  50  cc.  of  fresh  milk  add  10  cc.  of  N. 
calcium  chloride  (5-55  per  cent.),  and  make  the  volume  up  to  100  cc. 
with  distilled  water. 

Method.  Measure  5  cc.  of  the  calcined  milk  into  4  or  5  rather 
wide  test-tubes  and  place  them  in  the  warm  bath  at  38°  C.  Add 
i  cc.  of  the  enzyme  solution  to  one  of  the  tubes,  mix  by  placing  the 
thumb  on  the  top  of  the  tube  and  giving  one  shake ;  immediately 
replace  the  tube  in  the  bath  and  note  the  time.  Examine  the  tube 
at  intervals,  and  note  the  time  when  a  precipitate  appears.  With  a 
0-5  per  cent,  solution  of  commercial  pepsin  it  is  almost  instantaneous. 
If  the  clotting  time  is  very  short,  dilute  i  cc.  of  the  enzyme  with  9  or 
19  cc.  of  distilled  water ;  mix  well,  wash  out  the  pipette,  and  repeat 
the  test.  Continue  to  make  further  dilutions  of  the  solution  thus 
obtained  until  the  clotting  time  is  between  ij  and  if  minutes. 

Calculation.  The  suggested  unit  is  the  amount  of  pepsin  that 
will  clot  5  cc.  of  the  calcined  milk  in  100  seconds.  The  clotting  time 
is  inversely  proportional  to  the  amount  of  enzyme  present. 

Thus,  suppose  i  cc.  of  a  i  in  120  dilution  clots  in  85  seconds, 
then  i  cc.  of  the  original  solution  contains 

100 
120  x  -  -  =  141  units. 

85 

NOTE. — It  is  convenient  to  use  a  deep  water  bath  with  the  bulb  of  an 
electric  lamp  dipping  into  the  water.  The  tubes  are  contained  in  a  wire  basket 
fastened  to  the  inner  side  of  the  bath.  After  the  enzyme  has  been  added,  the 
tube  is  held  in  a  sloping  position  and  gently  rotated  backwards  and  forwards. 
The  formation  of  the  precipitate  can  be  readily  detected  in  the  light_from_the 


CH.  VIII.]  RENNIN.  207 

lamp.  In  this  way  the  temperature  of  the  mixture  can  be  maintained  con- 
stant, an  impossibility  when  the  tube  has  to  be  repeatedly  removed  for  in- 
spection. A  stop-watch  is  an  added  convenience. 


E.    Reniiin  and  the  Clotting  of  Milk. 

When  warm  milk  is  treated  with  a  neutral  or  faintly 
acid  extract  of  the  mucous  membrane  of  the  stomach  a 
clot  forms  after  a  certain  time.  The  solid  portion  or  curd 
contains  the  fat  held  together  by  insoluble  calcium  para- 
caseinate,  which  is  formed  from  the  soluble  calcium 
caseinate  of  the  milk  by  ferment  action.  The  fluid  portion 
or  whey  contains  the  lactalbumin,  lactose,  and  inorganic 
constituents. 

The  nature  of  the  enzyme  responsible  for  the  change 
has  been  much  discussed.  The  most  probable  view  is  that 
in  the  stomach  of  infants  and  other  sucklings  a  specific 
enzyme  called  rennin  is  present.  But  it  also  seems  to  be 
true  that  all  enzymes  that  can  hydrolyse  casein  can  cause 
milk  to  clot.  Thus  pepsin,  trypsin,  erepsin  and  many  of 
the  proteolytic  enzymes  found  in  plants  will  clot  milk  in 
the  presence  of  a  suitable  concentration  of  calcium  and  of 
hydrogen  ions.  Many  workers,  notably  Pawlow,  have 
urged  that  the  clotting  of  milk  by  extracts  of  the  stomach 
is  due  to  pepsin  alone,  and  that  a  specific  rennetic  enzyme 
does  not  exist.  Such  results  are  probably  due  to  the  fact 
that  they  only  studied  extracts  of  the  stomach  of  dogs  and 
pigs,  which  do  not^seem  to  contain  rennin.  The  results 
obtained  by  Bang  and  Hammersten  on  the  comparison 
of  the  various  enzymatic  activities  of  extracts  of  the  gastric 
mucosa  of  the  calf  and  the  pig  are  taken  by  the  author  as 
conclusive  evidence  of  the  existence  of  two  separate 
enzymes.  The  results  obtained  by  the  author  on  the 
relative  heat  destruction  of  the  clotting  powers  of  the  two 
extracts  at  a  definite  hydrogen-ion  concentration  can  be 
readily  repeated,  and  are  most  convincing.  (See  Ex.  256.) 


2C>8  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

The  main  differences  between  the  pepsin  and  rennin 
as  enzymes  that  can  clot  milk  are  : 

(1)  Pepsin    is    more    stimulated    by    increasing    the 
concentration  of  calcium  chloride  than  is  rennin. 

(2)  Pepsin  is  almost  completely  destroyed  by  heating 
for   10  minutes  at  38°  C.  at  PH  =  7-25.     Rennin 
only  loses  a  small  fraction  of  its  activity. 

(3)  Heating  for  2  minutes  at  70°  C.  at  PH  =  5  com- 
pletely   destroys    rennin,    but    has    no    effect    on 
pepsin.     Since  the  clotting  power   and   ordinary 
peptic  action  of  such  an  heated  solution  is  the 
same  as  the  unheated  solution,  it  must  be  con- 
cluded that  the  clotting  action  of  pepsin  is  due  to 
this  enzyme,  and  not  to  another  enzyme  associated 
with  pepsin.     This  is  in  opposition  to  the  view  of 
Bang,  who  claims  that  a  special  clotting  enzyme, 
which  he  calls  parachymosin,   is  found  in  pigs' 
stomach. 

The  remarkable  fact  that  pepsin  can  hydrolyse  casein  to 
paracasein  at  a  reaction  of  about  PH  =  6-7,  whilst  at  this 
reaction  it  does  not  act  on  any  other  protein,  has  not  been 
satisfactorily  explained. 

The  first  action  of  proteolytic  enzymes  on  casein  is  to 
hydrolyse  it  to  paracasein.  If  there  is  a  sufficient  con- 
centration of  calcium  ions  and  the  reaction  of  the  medium  is 
neither  too  acid  nor  too  alkaline,  the  paracasein  is  pre- 
cipitated as  the  insoluble  calcium  paracaseinate.  Pepsin 
in  markedly  acid  solution,  erepsin  in  solutions  that  are 
about  neutral  and  trypsin  in  neutral  and  faintly  alkaline 
solutions  can  hydrolyse  this  further  to  substances  allied  to 
the  proteoses  and  peptones  ("  caseoses  M),  and,  in  the  case 
of  erepsin  and  trypsin,  to  ammo-acids. 

Rennin  is  prepared  by  extracting  the  mucous  mem- 
brane of  the  fourth  stomach  of  a  sucking  calf  with  brine. 
It  can  be  obtained  commercially  in  the  solid  or  liquid  form, 
being  extensively  used  in  the  cheese  industry,  and  also  in 
the  kitchen  for  the  preparation  of  "  junket."  The  author 


CH.  VIII.]  RENNIN. 

has  found  that  certain  of  the  preparations  sold  recently 
contain  pepsin  rather  than  rennin,  due  to  the  fact  that  the 
shortage  of  calves'  stomachs  has  necessitated  the  use  of 
pigs'. 

253.  Measure  5  cc.  of  fresh  milk  into  a  tube  and  place  it  in  a 
water  bath  at  38°  C.  for  a  few  minutes.     Add  I  cc.  of  an  active 
preparation  of  rennet ;  mix,  replace  in  the  bath,  and  observe  from 
time  to  time.    Note  that  the  milk  sets  to  a  solid  mass,  so  that  the 
tube  can  be  inverted  without  spilling  the  contents.     Replace  the 
tube  in  the  bath  and  examine  it  again  after  about  an  hour.     Note 
that  the  clot  has  contracted,  and  that  a  nearly  clear  "  whey  "  has 
been  expressed. 

254.  Repeat  the  experiment  with  I  cc.  of  a  0-5  solution  of 
commercial  pepsin  in  water.     A  similar  result  is  obtained. 

255.  To  10  cc.  of  milk  add  3  cc.  of  0-2  N.  ammonium  oxalate 
to  remove  the  calcium  ions.     Mix,  and  divide  into  three  equal 
portions,  and  place  these  in  three  tubes,  labelled  A,  B  and  C. 

To  A  add  i  cc.  of  N.  calcium  chloride  and  i  cc,  of  rennin. 

To  B  add  i  cc.  of  rennin. 

To  C  add  i  cc.  of  boiled  rennin. 

Place  the  three  tubes  in  the  water  bath  for  10  minutes.  Note 
that  A  clots  and  that  B  and  C  do  not. 

Boil  B  (to  destroy  the  rennin)  and  cool  under  the  tap. 

To  B  and  C  add  i  cc.  of  N.  calcium  chloride.  A  flocculent  pre- 
cipitate of  calcium  paracaseinate  is  produced  in  B,  indicating  that 
the  enzyme  has  acted  on  the  casein  in  the  absence  of  the  calcium 
salts,  but  that  calcium  is  necessary  for  the  formation  of  the  pre- 
cipitate. 

NOTE. — If  the  experiment  be  repeated  with  pepsin  it  will  be  found  that  on 
boiling  B  and  adding  CaClg  little  or  no  effect  is  produced.  This  is  due  to  the 
fact  that  pepsin  only  seems  to  act  on  casein  In  the  presence  of  calcium  salts. 

256.  Distinction  between  Rennin  and  Pepsin. 

To  10  cc.  of  0-5  per  cent,  pepsin  in  water  add  5  cc.  of  0-2  M.  acid 
potassium  phosphate,  and  make  the  volume  up  to  TOO  cc.  with 
distilled  water.  Titrate  20  cc.  of  the  mixture  with  o-i  N.  soda  to  a 

p 


2IO  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

reaction  of  PH  =  7-25,  using  the  method  described  on  page  277, 
note  3.     Suppose  that  1-5  cc.  of  the  soda  are  required. 

To  10  cc.  of  the  diluted  enzyme  add  half  this  amount  of  o-i  N- 
soda,  mix  and  place  the  tube  in  the  warm  bath  for  10  minutes.  At 
the  end  of  the  heating  period  add  0-75  of  o-i  N.  HC1,  mix  and  cool 
under  the  tap.  Label  the  tube  A. 

To  another  10  cc.  of  the  diluted  enzyme  add  0-75  cc.  of  o-i  N. 
HC1,  and  then  0-75  cc.  of  o-i  N.  soda.  Mix,  and  label  the  tube  B. 
Determine  the  relative  clotting  powers  of  these  two  solutions  on 
calcined  milk  as  in  Ex.  252.  It  will  be  found  that  A  has  almost  lost 
its  action  as  compared  with  B. 

Perform  a  similar  experiment  with  a  solution  of  rennin,  adding 
the  phosphate  as  a  buffer,  diluting  to  100  cc.  and  determining  how 
much  soda  is  required  to  bring  it  to  PH  =  7-25.  Label  the  heated 
rennin  C  and  the  control  D.  On  comparing  their  clotting  powers 
as  before  it  will  be  found  that  heating  at  PH  =  7-25  has  compara- 
tively little  effect  on  rennin  as  compared  with  pepsin,  the  clotting 
power  usually  being  about  half  that  of  the  control. 

NOTES. — i .  In  class  work  it  is  convenient  for  the  Demonstrator  previously 
to  determine  the  amounts  of  soda  and  acid  required  for  10  cc.  of  the  two 
enzymes.  Otherwise  the  experiment  takes  a  considerable  time. 

2.  If  the  reaction  be  more  alkaline  than  PH  =  7-25  the  rennin  is  rapidly 
destroyed. 

3.  The  following  is  the  result  of  a  typical  experiment,  the  units  being 
calculated  as  explained  in  Ex.  252. 


Pepsin. 

Rennin. 

Control            

21-2 

23-1 

Heated  at  PH  =  7-25  for  10  mins.  .  . 

0-03 

11-4 

F.    Trypsin. 

Trypsin  is  the  proteolytic  enzyme  formed  by  the 
interaction  of  trypsinogen  and  enterokinase.  Trypsinogen 
is  elaborated  in  the  pancreas,  and  is  found  as  such  in  fresh 
pancreatic  juice.  Enterokinase  is  secreted  by  the  small 
intestine,  but  is  also  found  in  nearly  all  the  tissues  of  the 


CH.  VIII.]  TRYPSIN.  211 

body  in  small  amounts.  It  converts  trypsinogen  to 
trypsin,  but  the  mechanism  of  this  change  is  not  fully 
understood.  From  the  fact  that  a  small  amount  of  the 
kinase  can  in  time  activate  a  large  amount  of  trypsinogen 
it  is  probable  that  the  process  is  enzymatic,  and  not  merely 
the  union  of  two  substances,  each  of  which  alone  is  in- 
active. It  is  remarkable  that  the  rate  of  activation  is  at 
first  slow,  but  increases  very  rapidly  as  the  process  nears 
completion.  This  is  opposed  to  the  rate  of  action  of  other 
enzymes  which  is  most  rapid  at  the  commencement,  and 
which  falls  off  as  the  concentration  of  the  substrate 
decreases.  No  satisfactory  explanation  of  this  so-called 
"autocatalysis"  has  been  offered.  Enterokinase  acts  best 
in  a  faintly  acid  medium,  but  the  optimum  PH  has  not  been 
studied.  Since  the  contents  of  the  small  intestine  are  still 
acid  when  the  pancreatic  juice  is  secreted,  it  is  possible 
that  the  conditions  of  the  medium  are  such  as  will  enable 
the  enterokinase  to  rapidly  activate  the  trypsinogen. 

Trypsin  acts  best  in  a  slightly  alkaline  medium,  the 
optimum  PH  being  about  8-1.  In  the  absence  of  proteins, 
which  exert  a  protective  action,  it  is  slowly  destroyed  by 
alkalies  at  body  temperature.  At  room  temperature, 
according  to  the  author's  observations,  it  is  most  stable  at 
about  PH  =  5-5.  It  is  not  readily  destroyed  in  solutions  as 
acid  as  PH  =  i  *5,  though  at  such  a  reaction  it  does  not  act 
as  an  enzyme. 

According  to  the  observations  of  J.  Mellanby  and 
Wooley,  trypsin  is  more  stable  in  acid  solution  than  when 
neutral  or  alkaline.  They  claim  that  in  the  presence  of  a 
small  amount  of  free  acid  trypsin  can  be  heated  to  100°  C. 
for  5  minutes  without  being  completely  destroyed,  whereas 
in  slightly  alkaline  solution  it  is  completely  destroyed  at 
60°  C.  They  find  that  the  chlorides  of  barium  and  calcium 
are  effective  agents  in  preserving  trypsin  at  body  tempera- 
ture. 

Trypsin  acts  on  all  soluble  and  on  many  insoluble 
proteins.  It  finally  converts  them  to  a  mixture  of  amino- 
acids  and  of  relatively  simple  polypeptides.  The  hydrolysis 


212  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

of  the  protein  is  not  complete  even  with  a  very  prolonged 
period  of  digestion.  It  is  much  more  complete  if  the  pro- 
tein has  been  previously  submitted  to  peptic  digestion, 
indicating  the  presence  of  certain  groupings  in  the  protein 
molecule  that  are  attacked  by  pepsin,  and  not  by  trypsin. 
It  must  be  remembered  that  in  the  course  of  natural  diges- 
tion in  the  body  the  proteins  are  liable  to  the  attack  of 
three  proteolytic  enzymes,  pepsin,  trypsin  and  erepsin,  and 
that  it  is  possible  that  complete  hydrolysis  is  most  effec- 
tively and  rapidly  attained  if  the  three  enzymes  are  pre- 
sented in  due  order. 

The  course  of  hydrolysis  can  be  followed  by  Sorensen's 
method  of  formal  titration,  or  by  the  direct  estimation  of 
the  amino-acid  nitrogen  by  the  gasometric  method  of  D. 
Van  Slyke.  As  stated  above,  it  is  most  rapid  at  the  com- 
mencement of  the  reaction,  falling  off  as  the  hydrolysis 
reduces  the  concentration  of  the  substrate.  The  process 
is  a  very  complicated  one  to  follow  mathematically,  owing 
to  the  variety  of  substances  simultaneously  present  which 
are  liable  to  be  attacked  by  the  enzyme,  and  to  the  fact 
that  the  amount  of  enzyme  is  continuously  decreasing 
owing  to  its  instability.  For  the  estimation  of  trypsin 
it  is  preferable  to  rely  on  the  method  given  in  Ex.  259. 

Preparation  of  Trypsin  from  Pig's  Pancreas. 

The  following  method  is  a  modification  of  that  of  Mellanby  and  Wooley. 
It  must  be  noted  that  the  extract  obtained  does  not  contain  amylopsin  or 
lipase,  both  of  which  are  rapidly  destroyed  by  acids. 

Obtain  the  fresh  pancreas  of  the  pig  (usually  sold  as  the  "  internal  sweet- 
bread ").  Free  the  glandular  tissue  from  fat  as  completely  as  possible,  mince 
finely  in  a  machine  and  weigh.  For  every  gram,  of  the  mince  add  3  cc.  of 
0-5  per  cent,  hydrochloric  acid  (by  weight).  This  can  be  prepared  with 
sufficient  accuracy  by  diluting  13-7  cc.  of  pure  concentrated  hydrochloric 
acid  (Sp.  Gr.  1-16)  to  make  1000  cc.  with  distilled  water.  Stir  the  mixture 
well  at  intervals  for  30  minutes.  Then  add  6-4  cc.  of  5  per  cent,  soda  (or  8  cc. 
of  N.  soda)  for  every  100  cc.  of  the  dilute  hydrochloric  acid  originally  used. 
This  gives  a  reaction  of  about  PH  =  4-7,  which  results  in  a  readily  filterable 
mass.  Stir  thoroughly  and  filter  on  a  large  folded  paper.  To  the  filtrate, 
which  is  usually  quite  clear,  add  10  per  cent,  soda  to  reduce  the  acidity  to 
about  PH  =  5-5,  the  optimum  reaction  for  the  preservation  of  trypsin.  This 
can  be  obtained  approximately  by  cautiously  adding  the  soda  until  a  2  cc. 
portion  of  the  mixture  gives  only  a  faint  reddish  tinge  with  a  few  drops  of 
methyl  red.  Then  add  toluol  (10  cc.  per  litre)  as  a  preservative,  shake  and 
store  in  a  stoppered  bottle  in  a  cool,  dark,  cupboard.  Should  the  bottle  be 


CH.  VIII.]  TRYPSIN.  213 

opened,  it  may  be  necessary  to  add  a  little  more  toluol  and  to  shake  well  before 
returning  it  to  store. 

Trypsin  can  also  be  obtained  by  extraction  with  dilute  alcohol.  The 
material  left  over  after  the  preparation  of  lipase  (see  p.  158)  should  be  filtered. 
The  nitrate  contains  amylopsin  and  trypsin.  The  trypsin  is  more  permanent 
if  i  cc.  of  pure  concentrated  hydrochloric  acid  be  added  for  every  litre.  If 
amylopsin  is  required  the  acid  should  not  be  added. 

For  the  following  experiments  a  i  in  5  or  even  i  in  10 
dilution  of  the  above  extract  can  be  used. 

257.  Detection  of  Trypsin  by  the  use  of  Calcified  Milk. 

Measure  5  cc.  of  the  prepared  milk  (see  Ex.  252)  into  two  test- 
tubes  labelled  A  and  B,  and  place  them  in  a  water  bath  at  37°  C. 
To  A  add  i  cc.  of  the  solution.  Boil  a  little  of  the  solution  and  add 
i  cc.  of  the  cooled  solution  to  B,  using  a  clean  pipette.  Mix  the 
contents  of  the  tubes  separately,  replace  them  in  the  bath  and 
observe  at  intervals.  If  trypsin  is  present  the  milk  in  A  will  be 
precipitated  or  form  a  clot.  The  observation  is  valueless  if  the  milk 
in  B  also  clots,  as  may  happen  with  milk  which  has  "  turned  sour," 
or  even  with  fresh  milk  if  the  fluid  tested  is  very  acid. 

NOTE. — A  positive  result  indicates  the  presence  of  trypsin,  pepsin,  rennin, 
or  other  proteolytic  enzyme.  Further  tests  must  be  applied  (Ex.  248,  256, 
and  258).  But  it  may  be  noted  that  trypsin  will  cause  the  clot  to  disappear 
gradually,  a  phenomenon  not  obtained  with  the  other  enzymes. 

258.  Detection  of  Trypsin  by  the  use  of  casein  solution. 

To  10  cc.  of  the  casein  solution  (see  below)  add  2  cc.  of  the  enzyme 
solution,  mix,  label  the  tube  C,  and  place  it  in  the  water  bath  at  37 °C. 
Boil  a  little  of  the  enzyme  solution,  cool,  and  add  i  cc.  to  5  cc.  of  the 
casein.  Label  the  tube  D.  At  intervals  of  about  10  minutes 
transfer  about  i  cc.  of  C  to  a  tube,  and  add  i  per  cent,  acetic  acid, 
drop  by  drop.  If  trypsin  is  present,  it  will  be  found  that  after  a 
certain  interval,  depending  on  the  strength  of  the  ferment,  it  is  not 
possible  to  produce  a  precipitate  by  the  addition  of  acetic  acid. 
Should  this  stage  be  reached,  confirm  the  result  by  obtaining  a 
precipitate  of  casein  in  D  by  careful  acidification. 

NOTES. — i.  Casein  solution.  Weigh  out  i  gram,  of  Hammersten's  Casein 
(which  can  be  obtained  from  Casein,  Ltd.,  Battersea,  London,  S.W.)  into  a 
clean,  dry  beaker.  Add  about  20  cc.  of  distilled  water  and  stir.  Add  10  cc. 
of  o-i  N.  soda  and  stir  well.  Allow  to  stand  for  about  30  minutes,  and  make 
the  volume  up  to  TOO  cc.  with  distilled  water. 

2.  Casein  is  hydrolysed  by  trypsin  in  alkaline  neutral  or  faintly  acid 
solution.  The  first  product  of  hydrolysis  is  paracasein,  which  is  precipitated 


214  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

as  calcium  paracaseinate  if  there  be  a  sufficient  concentration  of  calcium  salts 
present,  as  there  is  in  Ex.  257.  The  later  products  of  hydrolysis  are  bodies 
allied  to  the  proteoses  (caseoses),  peptones,  and  finally  the  ammo-acids.  The 
failure  to  obtain  a  precipitate  with  acetic  acid  marks  the  stage  when  the  last 
trace  of  paracasein  has  been  hydrolysed.  Though  rennin  and  pepsin  in 
neutral  or  faintly  acid  solution  can  hydrolyse  casein  to  paracasein,  they  are 
unable  to  effect  the  further  stages  of  break  down,  and  therefore  do  not  give 
this  important  test  for  casein. 

259.  Estimation  of  Trypsin.      Having  demonstrated  that  a 
given  solution  contains  trypsin,  this  can  be  estimated  by  following 
the  procedure  given  in  Ex.  252,  as  first  suggested  by  J.  Mellanby. 
The  same  unit  as  that  adopted  for  pepsin  is  convenient. 

260.  The  course  of  tryptic  digestion  of  casein  as  followed  by 
formol  titrations. 

Principle  of  the  method.     Suppose  the  constitution  of  a  protein  to  be 
represented  by  the  following  formula  : 

R  Rx  R2  R3 


I  I  I 

.CH. 


H2N.CH.CO  -  NH.CH.CO  -  NH.CH.CO  -  NH.CH.COOH. 

The  aqueous  solution  of  such  a  compound  would  probably  be  nearly  neutral, 
the  acidity  due  to  the  terminal  -  COOH  group  being  balanced  by  the  basicity 
due  to  the  terminal  -  NH2.  The  addition  of  a  small  amount  of  soda  would 
render  the  solution  alkaline  to  phenol  phthalein.  On  adding  a  neutralised 
solution  of  commercial  formaldehyde  (formol)  the  basicity  of  the  -  NH2 
group  is  removed  by  the  formation  of  a  methylene  group  [see  p.  69  (3)]. 

-  NH2  +  OHC.H  =  -  N  :  CH2  +  H.,0. 

The  whole  molecule  would  now  behave  as  an  acid  owing  to  the  unopposed 
influence  of  the  terminal  -  COOH  group  ;  and  the  solution  would  require  the 
addition  of  an  equivalent  of  soda  to  make  it  again  alkaline  to  phenol  phthalein. 
Let  this  amount  of  soda  be  A. 

If  the  protein  be  hydrolysed  by  an  enzyme  into  its  constituent  amino- 
acids,  the  amount  of  soda  required  for  neutralisation  would  probably  be 
approximately  the  same  as  that  for  the  intact  protein.  But  after  treatment 
with  formol  the  amount  required  to  make  the  solution  alkaline  to  phenol 
phthalein  would  be  four  times  greater  than  A.  The  reason  for  this  is  that  after 
hydrolysis  there  are  four  free  amino-groups  and  four  free  carboxylic  groups, 
and  the  solution  is  still  about  neutral.  But  after  treatment  with  formol  the 
basic  influence  of  all  four  amino  groups  is  removed,  and  to  make  the  solution 
alkaline  enough  soda  has  to  be  added  to  neutralise  all  four  carboxylic  groups. 
It  therefore  follows  that  the  degree  of  hydrolysis  of  such  a  protein  can  be 
followed  by  determining  the  amount  of  standard  alkali  required  to  neutralise 
the  solution  after  treatment  with  formol. 

The  usual  method  adopted  is  to  withdraw  samples  of  the  digesting 
mixture  at  intervals,  add  neutralised  formol  and  titrate  to  a  definite  pink  colour 
to  phenol  phthalein.  The  amount  required  (less  the  amount  for  a  sample 
taken  immediately  after  the  addition  of  the  enzyme)  is  a  measure  of  the  number 
of  amino-groups  set  free.  In  some  cases  the  solution  is  neutralised  to  litmus 
before  the  addition  of  the  formol.  A  practical  difficulty  is  that  of  defining  the 
exact  end  point  of  the  titration  so  that  the  same  final  reaction  is  obtained  in 


CH.  VIII.]  FORMOL  TITRATION.  215 

the  control  and  in  all  the  observations  made.  The  digestion  mixture  is  usually 
opalescent  and  pigmented.  A  simple  control  is  obtained  by  boiling  a  portion 
of  the  digest  in  a  flask  to  destroy  the  enzyme,  adding  formol  and  phenol 
phthalein  and  titrating  to  a  definite  pink.  The  control  and  all  subsequent 
titrations  are  brought  to  the  same  colour.  But  the  best  results  are  obtained 
by  use  of  the  comparator  of  Cole  and  Onslow,  constructed  to  hold  large  boiling 
tub  (see  fig.  37).  With  this  it  is  possible  to  titrate  sharply  to  a  definite  PH. 
The  principle  underlying  the  method  of  titration  is  described  in  Ex.  322.  To 
save  material  in  this  experiment  only  one  buffer  solution  is  used,  instead  of 
the  two  in  Ex.  322. 

A  further  point  of  interest  in  connexion  with  formol  titrations  is  the 
fact  that  the  amount  of  soda  required  to  make  the  digestion  mixture  alkaline 
to  PH  =  8-3  (pink  with  phenol  phthalein)  increases  during  the  course  of  diges- 
tion, being  especially  marked  in  the  early  stages  of  tryptic  digestion.  This  is 
not  observed  in  the  experiment  conducted  by  the  method  described  below, 
owing  to  the  fact  that  the  formol  is  added  directly  to  the  sample.  The  rela- 
tionships between  the  amounts  of  alkali  required  before  and  after  the  addition 
of  formol  with  various  proteins  subjected  to  the  action  of  different  proteolytic 
enzymes  is  being  carefully  studied,  as  it  seems  possible  that  they  will  throw 
light  on  the  nature  of  the  groupings  in  the  protein  molecule  that  are  liable 
to  attack  by  the  different  enzymes. 

Formol  Solution.  Dilute  commercial  formaldehyde  (40  per  cent.)  with 
an  equal  volume  of  distilled  water.  Add  10  drops  of  a  0-5  per  cent,  solution  of 
phenol  phthalein  in  50  per  cent,  alcohol  for  every  100  cc.  of  the  solution  and 
titrate  with  o-i  N.  soda  until  a  faint  pink  tinge  is  obtained.  It  may  be 
necessary  to  add  a  few  more  drops  of  the  alkali  from  time  to  time  owing  to  the 
oxidation  of  the  formaldehyde  to  formic  acid. 

Casein  Solution.  Make  a  10  per  cent,  solution  of  commercial  casein  accord- 
ing to  the  directions  given  in  Ex.  87  A  (i.)  to  (vi.).  Add  toluol,  shake  well, 
and  place  in  a  deep  water  bath  or  air  incubator  at  38°  to  40°  C.  for  24  hours, 
shaking  at  intervals.  When  all  preparations  for  the  titrations  have  been 
made,  there  are  added  25  to  50  cc.  per  litre  of  the  pancreatic  extract  described 
on  p.  212.  The  bottle  is  well  shaken,  a  sample  immediately  withdrawn  for 
titration,  and  the  bottle  replaced  in  the  incubator  or  water  bath.  Further 
samples  are  taken  at  intervals,  the  following  making  a  suitable  series  :  £,  \,  i, 
2,  6,  24  and  48  hours. 

Method  of  titration.  Withdraw  20  cc.  of  the  digestion  mixture 
with  a  pipette  and  transfer  it  to  the  tube  (3)  of  the  comparator 
(fig.  37).  Another  portion  of  20  cc.  is  placed  in  tube  (2).  In  (i) 
place  25  .cc.  of  a  buffer  solution  of  PH  =  8-5  (see  p.  28).  In  (4)  place 
about  25  cc.  of  water.  Into  tubes  (i)  and  (3)  measure  10  drops  of 
O'5  per  cent,  phenol  phthalein  by  means  of  a  dropping  pipette 
(fig.  5).  To  (3)  add  5  cc.  of  the  neutralised  formol.  To  (2)  add  5  cc. 
of  water  to  make  the  colour  and  opalescence  comparable  with  that 
of  (3).  Titrate  (3)  with  0-2  N.  soda  from  a  burette.  The  end  point 
is  reached  when  the  appearance  seen  on  the  ground  glass  screen  at 
Y  is  the  same  as  that  seen  at  X.  If  more  than  2  cc.  of  the  alkali  are 
required,  the  same  volume  of  water  should  be  added  to  tubes  (i)  and 


2l6  COMPOSITION    OF   THE   DIGESTIVE   JUICES.  [CH.  VIIl. 

(2).  Thus,  suppose  that  just  before  the  end  point  is  reached  4-5  cc. 
of  the  0-2  N.  soda  have  been  added  to  (3).  Add  4-5  cc.  of  water  to 
(i)  and  to  (2),  and  then  complete  the  titration  till  exact  equality  of 
tint  is  obtained. 

Calculation. 

1000  cc.  N.  soda  =*  14  grams,  of  amino-acid  nitrogen, 

i  cc.  of  0-2  N.  soda  =2-8  mgm.  amino-acid  nitrogen. 

If  (a)  =  amount  of  0-2  N.  soda  required  for  20  cc.  of  the 
digestion  mixture  immediate!}'  after  the  addition  of  the  enzyme, 
and  (b)  the  amount  after  an  interval  of  (t)  minutes,  then 
[(b)  —  (a)]  x  5  x  2-8  =  mgms.  of  amino-acid  nitrogen  liberated  in 
100  cc.  of  the  digestion  mixture  in  (t)  minutes. 

It  will  be  found  that  the  rate  of  digestion,  i.e.  the  mgms.  of 
amino-acid  nitrogen  liberated  in  i  minute,  is  greatest  at  the  com- 
mencement of  the  digestion,  falling  off  as  the  concentration  of  the 
substrate  decreases. 

NOTES. — i.  If  a  parallel  experiment  be  conducted  with  chloroform 
instead  of  toluol  as  the  antiseptic,  it  will  be  found  that  the  digestion  rate  is 
considerably  less.  For  that  reason  it  is  preferable  to  use  toluol  instead  of 
chloroform  for  the  preservation  of  trypsin  solutions  and  as  an  antiseptic  in 
digestions. 

2.  The  percentage  amount  of  the  casein  digested  is  much  greater  in 
dilute  than  in  strong  solution.  The  addition  of  an  equal  volume  of  water  will 
induce  a  greater  degree  of  hydrolysis  in  the  above  experiment  than  doubling 
the  amount  of  enzyme. 

261.    The  products  of  tryptic  digestion  of  casein. 

The  final  products  are  given  on  pages  67  to  69.  The  methods 
of  separation  are  discussed  on  p.  70,  and  the  details  of  the  special 
methods  required  for  the  isolation  of  tryptophane,  tyrosine  and 
leucine  are  given  in  Exs.  87,  89  and  93  respectively.  The  following 
exercise  is  inserted  for  the  benefit  of  junior  students  as  illustrating 
the  principles  of  the  methods  used. 

Digestion.  The  solution  and  digestion  is  carried  out  as  de- 
scribed in  Ex.  87  A  (i.)  to  (x.),  but  the  digestion  period  may  with 
advantage  be  extended  to  about  2  weeks.  The  bottle  is  removed 
from  the  incubator,  and  the  contents  transferred  to  a  3  litre  flask, 
and  heated  on  a  boiling  water  bath  or  in  a  steamer  for  about  i  hour. 
The  mixture  is  then  filtered  hot  and  a  portion  evaporated  in  the 


OH.  Vlll.j  TfcYPTlC  DIGESTION.  217 

boiling  water  bath  to  about  half  its  bulk.  This  is  distributed  into 
small  beakers  (labelled  B)  and  allowed  to  stand  in  a  cool  place  for 
24  hours  for  the  tyrosine  to  separate  out.  The  remaining  portion 
is  distributed  into  test-tubes  (labelled  A)  and  allowed  to  stand  for 
24  hours. 

(i.)  Bromine  reaction  for  free  tryptophane.  If  necessary  filter  A 
from  a  crystalline  precipitate  of  tyrosine.  Acidify  about  5  cc.  with 
a  couple  of  drops  of  strong  acetic  acid  and  add  bromine  water,  drop 
by  drop ;  a  pink  colour  gradually  develops,  which  deepens  and  then 
disappears  as  more  bromine  water  is  added.  When  the  colour  is 
no  longer  intensified  by  the  addition  of  bromine,  add  2  or  3  cc.  of 
amyl  or  butyl  alcohol  and  shake.  On  standing,  the  alcohol  rises 
to  the  surface  coloured  a  fine  red  or  violet.  (See  p.  93.) 

(ii.)  Treat  another  5  cc.  of  the  filtrate  with  I  cc.  of  25  per  cent, 
sulphuric  acid  and  10  cc.  of  a  10  per  cent,  solution  of  mercuric 
sulphate  in  5  per  cent.  H2SO4.  Shake  the  tube  and  leave  it  for  10 
minutes.  Note  the  yellow  precipitate  of  a  mercury  compound  of 
tryptophane.  Filter  this  off  and  label  the  filtrate  C.  Wash  the 
precipitate  through  a  hole  in  the  paper  into  a  clean  tube,  fill  with 
water,  shake  and  filter  again,  neglecting  the  filtrate.  Wash  the 
precipitate  on  the  paper  once  more  with  water  and  then  let  it  drain. 
Scrape  a  portion  off  the  paper,  transfer  it  to  a  tube,  add  2  cc.  of 
"  glyoxylic  reagent  "  and  then  2  cc.  of  concentrated  sulphuric  acid. 
A  purple  colour  is  produced,  showing  that  tryptophane  is  responsible 
for  the  glyoxylic  reaction.  (See  Ex.  23.) 

Treat  another  portion  of  the  precipitate  with  Millon's  reagent 
and  boil.  A  yellow  colour  is  produced,  not  the  characteristic  red  of 
Millon's  reaction. 

To  another  portion  of  the  precipitate  apply  the  xanthoproteic 
test.  A  well-marked  reaction  is  obtained.  (See  Notes  to  Ex.  21.) 

To  portions  of  filtrate  C  apply  the  glyoxylic,  Millon's  and  the 
xanthoproteic  reactions.  Only  the  latter  two  are  obtained,  the 
trytophane,  but  not  the  tyrosine,  having  been  removed  by  the 
mercury  reagent  employed.  It  will  be  noted  that  the  solution 
becomes  bright  pink  as  soon  as  the  Millon's  reagent  is  added.  This  is 
due  to  the  influence  of  the  sulphuric  acid.  On  heating  the  colour 
may  be  discharged  if  an  excess  of  the  reagent  be  used, 


COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [cH.  VIII. 

(iii.)  Examine  the  crystalline  deposit  in  A,  or,  failing  this,  the 
mass  in  B.  Under  the  lower  power  of  the  microscope,  tyrosine  will 
appear  as  sheaves  or  fan-shaped  aggregates  of  needles.  The  material 
in  B  may  also  contain  spheres  or  cones  with  a  radiating  striation 
consisting  of  leucine.  Should  these  not  be  found,  the  crystalline 
mass  should  be  filtered  off  by  use  of  a  suction  pump  and  the  filtrate 
concentrated  on  a  boiling  water  bath.  On  standing  for  24  hours 
the  characteristic  leucine  balls  will  separate  out. 

G.     Erepsin. 

This  is  a  proteolytic  enzyme  widely  distributed  in  the 
animal  and  vegetable  kingdoms.  In  animals  it  is  especially 
abundant  in  the  mucous  membrane  of  the  intestine, 
particularly  in  the  jejunum.  It  is  found  in  the  succus 
entericus. 

It  differs  from  trypsin  in  that  it  has  no  action  on  such 
native  proteins  as  albumins  and  globulins.  But  it  hydro- 
lyses  the  proteoses  and  peptones  and  also  casein.  The 
final  products  of  action  are  the  same  as  in  the  case  of 
trypsin,  that  is,  free  amino-acids.  But  it  would  seem  that 
it  can  break  down  many  of  the  polypeptides  that  are 
resistant  to  the  action  of  trypsin.  Thus  the  products  of 
ereptic  digestion  may  fail  to  give  the  biuret  reaction, 
whereas  the  most  prolonged  tryptic  digests  give  a  vivid 
reaction  owing  to  the  polypeptides  present.  The  enzyme 
is  probably  of  great  importance  in  protein  digestion  in 
completing  the  hydrolysis  initiated  by  the  successive  action 
of  pepsin  and  trypsin.  It  is  usually  stated  that  the 
optimum  PH  for  the  action  of  erepsin  is  7-8. 

Preparation.  Obtain  a  length  of  the  small  intestine  of  a  recently  killed 
pig.  Wash  out  the  contents  under  the  tap,  split  open,  and  spread  on  a  board 
with  the  mucous  membrane  uppermost.  Scrape  this  off  the  muscle  coats  with 
the  back  of  a  scalpel,  placing  the  material  in  a  weighed  dish.  Determine  the 
weight  of  mucosa  obtained.  Grind  it  with  sand,  add  about  15  times  its  weight 
of  water  and  transfer  to  a  flask.  Add  some  toluol  and  shake.  After  standing 
for  about  half  an  hour  add  i  gram,  of  sodium  chloride  for  every  100  cc.  of  water 
taken,  shake  till  dissolved,  and  filter  through  a  pleated  paper.  Filtration  is 
very  slow,  and  may  take  several  hours.  It  can  be  accelerated  by  the  addition 
of  a  little  acetic  acid,  which  precipitates  some  of  the  mucin  and  nucleo-proteins 
which  make  the  solution  so  slimy.  There  is  a  risk  of  destroying  the  enzymes 
by  adding  too  much  acid,  but  in  some  cases  the  addition  of  minimal  amounts 


CH.  VIII.]  EREPSIN.  219 

gives  a  more  active  preparation.  Toluol  should  be  added  to  the  flask  in 
which  the  nitrate  is  collected.  The  various  enzymes  in  the  fluid  are  not  very 
stable,  so  that  the  material  must  be  obtained  and  the  extract  made  not  more 
than  two  days  before  it  is  required. 

262.  Action    of    erepsin   on  peptone.      Label  two  150   cc. 
flasks  A  and  B,  and  into  each  place  100  cc.  of  a  2  per  cent,  solution 
of  commercial  peptone.     To  A  add  10  cc.  of  the  filtered  extract  and 
2  or  3  cc.  of  toluol,  shake  well,  and  stopper  with  a  cork.     Boil 
another  10  cc.  of  the  extract  to  destroy  the  enzymes,  cool,  and  add  to 
B.     Add  tolnol  and  stopper.     Incubate  the  flasks  at  38°  to  40°  C. 
for  24  to  48  hours  or  longer. 

(i.)  To  5  cc.  of  each  add  two  or  three  drops  of  strong  acetic 
acid  and  then  bromine  water,  drop  by  drop  (see  Ex.  261).  A  pink 
colour  is  obtained  in  A,  but  not  in  B,  showing  that  the  tryptophane 
bound  in  the  peptones  has  been  set  free  by  the  action  of  a  ferment. 

(ii.)  Titrate  20  cc.  of  A  and  B  according  to  the  directions  given 
in  Ex.  260.  A  considerable  increase  in  amino-acid  nitrogen  results, 
owing  to  the  splitting  of  the  peptide  linkages  of  the  peptones  by  the 
action  of  erepsin. 

(iii.)  To  i  cc.  of  A  and  B  add  4  or  5  cc.  of  water,  2  drops  of  i 
per  cent,  copper  sulphate,  and  i  or  2  cc.  of  5  per  cent.  soda.  A 
strong  biuret  reaction  (Ex.  24)  is  given  by  B,  whereas  A  may  give 
none,  or  only  a  feeble  reaction. 

NOTE. — It  is  not  always  possible  to  obtain  a  solution  that  fails  to  give 
the  biuret  test.  It  seems  to  depend  on  the  quality  of  the  peptone 
employed. 

263.  Action   of  erepsin  on  casein.     Prepare  a  2-5  per  cent, 
solution  of  casein  in  dilute  alkali  by  diluting  i  part  of  the  solution 
described  in  Ex.  87  with  3  parts  of  water.     To  50  cc.  add  10  cc. 
of  the  intestinal  extract  and  toluol  and  label  A.     To  another  50  cc. 
add  10  cc.  of  water  (or  of  a  boiled  intestinal  extract),  tolvol,  and 
label  B.      Incubate  the  stoppered  flasks  for  2  to  7  days  at  38°  to  40°. 

To  portions  of  the  resulting  solutions  carefully  add  dilute  acetic 
acid,  drop  by  drop.  A  precipitate  of  unchanged  casein  is  pro- 
duced in  B.  In  A  the  precipitate  is  much  less  or  may  be  absent. 

To  the  acidified  solutions  thus  obtained  add  bromine  water, 
drop  by  drop.  A  reaction  for  free  tryptophane  is  produced  in  A, 
but  not  in  B. 


220  COMPOSITION   OF  THE   DIGESTIVE   JUICES.          [CH.  VIII. 

H.    Amylopsin. 

This  amylolytic  enzyme  is  secreted  by  the  pancreas. 
According  to  most  observers  it  has  an  action  identical  with 
that  of  ptyalin. 

J.  Mellanby  and  Wooley*  state  that  pancreatic  juice 
alone  converts  starch  to  stable  dextrin  (25  per  cent.)  and 
maltose  (75  per  cent.),  but  that  after  treatment  with  small 
amounts  of  acid  it  can  carry  the  hydrolysis  as  far  as  glucose, 
owing  to  the  appearance  of  maltase,  the  enzyme  which 
converts  maltose  to  glucose.  Since  the  pancreatic  juice 
on  entering  the  small  intestine  is  mixed  with  the  acid  chyme 
it  is  probable  that  the  observations  quoted  are  of  consider- 
able importance. 

Extracts  of  the  pig's  pancreas  normally  contain  maltase 
as  well  as  amylase.  The  enzymes  are  destroyed  by  acids, 
and  so  are  not  found  in  the  extract  described  on  page  212. 
According  to  Mellanby  and  Wooley  the  amylopsin  is  best 
obtained  by  extracting  the  minced  pancreas  with  twice  its 
weight  of  pure  glycerol  for  24  hours  at  37°  C. 

Preparation.  The  pancreas  is  extracted  with  dilute  alcohol  as  described 
on  p.  158.  After  standing  3  days  the  mass  is  filtered.  It  is  not  necessary  to 
add  a  preservative,  the  alcohol  acting  as  such.  The  amylase  and  maltase  are 
somewhat  unstable,  the  best  results  being  obtained  with  recently  prepared 
extracts. 

264.  The  action  of  amylopsin  on  starch.  To  20  cc.  of  a  3  per 
cent,  solution  of  soluble  starch  add  I  cc.  of  5  per  cent,  sodium 
chloride,  divide  into  two  equal  portions,  and  place  them  into  two 
test-tubes,  labelled  A  and  B.  To  A  add  I  cc.  of  the  pancreatic  extract 
and  a  few  drops  of  toluol.  Shake,  stopper,  and  incubate  for  24 
hours  at  38°  to  40°  C.  To  B  add  about  I  cc.  of  saliva,  then  tolvol, 
and  incubate  with  A. 

To  the  digested  fluids  add  phenyl-hydrazine  hydrochloride, 
sodium  acetate,  and  acetic  acid,  and  proceed  as  directed  in  Ex.  109. 
Examine  the  tubes  after  they  have  been  in  the  boiling  water  bath  for 
30  minutes.  A  will  probably  contain  a  precipitate  of  phenyl- 
glucosazone,  whilst  D  will  remain  clear  (see  Ex.  122).  Examine  a 


*  Journ.  of  Physiology,  xlix.,  p.  246. 


CH.  VIII.]  MALTASE,  ETC.  221 

portion  of  the  deposit  in  A  under  the  microscope  and  note  the 
characteristic  crystals  of  the  glucosazone.  In  B  the  sugar  produced 
is  maltose,  the  osazone  of  which  does  not  separate  in  the  boiling 
water  bath. 

265.  The  action  of  pancreatic  maltase.    Measure  10  cc.  of  a 
2  per  cent,  solution  of  maltose  into  two  tubes,  C  and  D.     To  C  add 
2  cc.  of  the  pancreatic  extract  and  a  little  toluol.     Boil  2  cc.  of  the 
extract,  cool  and  add  it  to  D,  together  with  a  little  toluol.     Stopper 
the  tubes  and  incubate  for  24  to  48  hours  at  38°  to  40°  C.     Prepare 
the  osazones  as  described  in  the  previous  exercise.     C  generally  gives 
a  fair  yield  of  glucosazone.     In  D  there  is  no  separation  whilst  hot, 
but  on  cooling  slowly  the  characteristic  crystals  of  maltosazone 
separate  out  (Ex.  122). 

I.    Maltase,  Lactase  and  Sucrase. 

These  enzymes  hydrolyse  the  three  chief  disaccharides 
into  their  constituent  monosaccharides.  They  are  all  found 
in  the  succus  entericus  and  the  mucous  membrane  of  the 
small  intestine.  Maltase  is  also  present  in  pancreatic 
extracts  (see  above). 

By  the  action  of  these  enzymes  the  sugars  of  the  food 
and  those  formed  by  the  action  of  ptyalin  and  amylopsin 
in  starch  are  converted  to  glucose,  fructose  and  galactose 
before  absorption.  The  disaccharides  themselves  are 
never  found  in  the  blood,  and  if  injected  they  are  rapidly 
excreted  in  the  urine. 

266.  Maltase.      To  50  cc.  of  a  2  per  cent,  solution  of  maltose 
add  10  cc.  of  the  extract  of  pig's  intestine  (see  p.  218)  and  a  little 
toluol.     Transfer  to  a  small  flask  labelled  E,  stopper,  shake  well,  and 
incubate  for  24  to  48  hours  (or  longer)  at  38°  C.     Boil  10  cc.  of  the 
extract  and  cool  under  the  tap.     Add  it  to  another  50  cc.  of  the 
maltose,  label  the  flask  F,  add  toluol,  and  incubate  as  a  control. 

With  10  cc.  of  E  and  F  proceed  to  prepare  the  osazones  as 
directed  in  the  previous  exercise.  E  generally  gives  a  good  yield  of 
glucosazone,  whilst  the  solution  is  still  hot,  whilst  F  only  yields  the 
maltosazone  on  cooling. 


222  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

The  remaining  50  cc.  of  the  solutions  may  be  treated  with  5  cc. 
of  the  A  mercuric  nitrate  solution  (p.  391)  and  filtered.  On  examining 
the  clear  filtrates  polarimetrically  it  will  be  found  that  the  rotation 
in  E  is  less  than  in  F,  owing  to  the  fact  that  the  specific  rotatory 
power  of  glucose  is  less  than  that  of  maltose. 

It  is  usually  possible  to  demonstrate  the  presence  of  glucose  in 
E  by  the  author's  method,  using  blood  charcoal.  Five  cc.  of  the 
two  solutions  are  diluted  with  15  cc.  of  water,  and  the  parallel  tests 
conducted  as  described  in  Ex.  381. 

267.  Lactase.     Repeat  the  above  exercise,  using  a  2  per  cent, 
solution  of  lactose  instead  of  the  maltose.     The  osazone  method 
usually  gives  a  definite  result,  as  does  the  method  with  blood  char- 
coal.    Polarimetric  observations  are  of  little  use  with  lactose,  owing 
to  the  fact  that  the  rotation  of  the  solution  is  only  slightly  changed 
by  the  hydrolysis  of  lactose. 

268.  Sucrase  (Invertase).    To  20  cc.  of  2  per  cent,  sucrose  add 
5  cc.  of  the  intestinal  extract  and  a  little  toluol ;    shake,  label, 
stopper  and  incubate  for  24  to  48  hours.    Perform  a  control  experi- 
ment by  first  boiling  the  extract. 

Examine  the  two  solutions  for  reducing  sugar,  which  appears 
as  the  result  of  the  action  of  the  enzyme. 

J.    Bacterial  decomposition  in  the  intestine. 

A  large  number  of  micro-organisms  are  common 
inhabitants  of  the  intestine,  being  especially  abundant  in 
the  colon.  Their  growth  and  activities  are  dependent  on  a 
variety  of  conditions,  such  as  reaction,  supply  of  food 
materials,  etc.  It  is  probable  that  the  changes  induced, 
even  by  a  specific  organism,  are  affected  by  the  action  of 
other  bacteria.  For  example,  the  fermentation  of  carbo- 
hydrates to  lactic  and  other  acids  by  certain  organisms 
may  inhibit  the  growth  or  modify  the  chemical  activities 
of  other  types. 

It  is  not  possible  to  give  here  a  full  account  of  the 
various  changes  brought  about  by  bacteria  in  the  intestine, 
but  certain  of  the  products  formed  by  the  putrefaction  of 


CH.  VIII.] 


BACTERIAL   DECOMPOSITION. 


223 


the  proteins  and  amino-acids  have  such  powerful  physio- 
logical actions  that  they  merit  special  attention. 

It  is  probable  that  undigested  protein  is  more  liable 
to  putrefaction  than  are  the  amino-acids  themselves,  since 
the  latter  are  rapidly  absorbed  from  the  small  intestine, 
where,  normally,  bacterial  action  is  not  very  pronounced. 
For  that  reason,  if  the  proteins  of  the  food  are  well  cooked, 
thoroughly  masticated,  and  readily  digested,  the  ill-effects 
due  to  the  absorption  of  the  decomposition  products  are 
not  likely  to  occur. 

The  changes  are  probably  due  to  the  action  of  enzymes 
secreted  by  the  growing  organisms.  But  such  enzymes  are 
not  easily  extracted  from  their  bodies.  The  order  in 
which  the  changes  occur  has  been  much  discussed.  Mr.  H. 
Raistrick  has  made  a  special  study  of  certain  of  the  reactions 
involved,  and  has  suggested  the  scheme  given  on  the  next 
page  as  indicating  the  lines  on  which  the  decomposition 
takes  place. 

The  following  list  gives  examples  of  the  substances 
corresponding  to-the  types  A  to  F  of  the  scheme,  which  are 
formed  by  the  bacterial  decomposition  of  tyrosine,  trypto- 
phane  and  histidine. 


Tyrosine. 

Tryptophane. 

Histidine. 

A.     Amines. 

(p  -  oxy  -  phenyl- 
ethyl  -  amine)  or 
Tyramine. 

Indol    ethyl 
amine. 

/?-iminazol-ethyl- 
amine  or  Hista- 
mine. 

B.     Unsaturated. 



Iminazol  acrylic 
acid  or  Urocanic 
acid. 

C.     Saturated 
Acid  I. 

p  -  oxy  -  phenyl- 
propionic  acid. 

Indol  propionic 
acid. 

Iminazol  propi- 
onic acid. 

D.     Saturated 
Acid  II. 

p  -  oxy  -  phenyl  - 
acetic  acid. 

Indol  acetic  acid. 



E. 

£-cresol. 

Scatol. 

F. 

Phenol. 

Indol. 

224 


COMPOSITION   OF  THE   DIGESTIVE   JUICES.          [CH.  VIII. 


a  ^ 
&^ 

II 


g  i 

w"  ^ 

O  a 
05 


M 


CO 

."2 


§1 

M      8 


S 


K 


03 


^  5 


11 

aT  ^ 

O      -tj 


^ 


c? 


CH.  VIII.]  BACTERIAL  DECOMPOSITION.  225 

The  amines  are  of  considerable  importance.  Even 
the  relatively  simple  compounds  mentioned  above  are  very 
active  physiologically,  causing  contraction  of  non-striped 
muscle,  which  generally  results  in  a  rise  of  blood  pressure. 
Histamine  is  a  very  powerful  drug,  and  causes  contraction 
of  the  uterus.  These  bases  are  present  in  ergot,  and  are 
produced  by  the  action  of  micro-organisms  on  the  proteins 
of  the  infected  rye.  The  physiological  activity  of  ergot  is 
mainly  due  to  the  presence  of  tyramine  and  histamine. 

In  addition  to  the  putrefaction  bases  mentioned  above, 
many  others  have  been  identified.  These  bases  are  some- 
times known  as  "  ptomaines." 

Putrescine,  NH2.CH2.CH2.CH2.CH2.NH2,  is  derived  from 
ornithine  by  the  loss  of  CO2.  Ornithine  itself  is  NH2.CH2. 
CH2.CH2.CH(NH2).COOH,  and  is  obtained  by  the  hydro- 
lysis of  arginine  (see  p.  69). 

Cadaverine,  NH2.CH2.CH2.CH2.CH2.CH2.NH2,  is  simi- 
larly formed  by  the  decarboxylation  of  lysine. 

A  complete  account  of  these  compounds  is  given  in 
Barger's  "  The  Simpler  Natural  Bases." 

Of  the  other  substances  mentioned,  urocanic  acid  was 
originally  isolated  from  the  urine  of  a  dog  ;  hence  its  name. 
Raistrick  has  recently  obtained  it  from  histidine  by  bacterial 
decomposition. 

Phenol  and  p-cresol  are  mentioned  again  on  p.  282. 

Indol  and  scatol  are  mainly  responsible  for  the  charac- 
teristic faecal  odour.  They  are  important  to  the  practical 
bacteriologist,  owing  to  the  fact  that  only  certain  organisms 
are  able  to  produce  them  from  tryptophane.  For  this 
reason  certain  suspected  organisms  are  grown  in  a  suitable 
medium,  which  is  tested  for  indol  after  a  given  time.  The 
information  thus  obtained  is  used  in  arriving  at  a  diagnosis 
of  the  organism.  The  usual  medium  used  is  a  solution 
of  Witte's  peptone,  and  the  incubation  period  is  2  to  8  days. 

Q 


226  COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 

Now  peptone  does  not  contain  free  tryptophane,  and  some 
specimens  of  peptone  only  contain  small  quantities  of 
combined  tryptophane,  as  can  be  determined  by  the 
failure  to  get  a  vivid  glyoxylic  reaction  (Ex.  23).  Before 
the  bacteria  can  make  indol  it  is  probable  that  they  have  to 
multiply  considerably  so  as  to  produce  the  various  enzymes 
concerned.  If  the  nutrient  medium  contains  free  trypto- 
phane and  is  otherwise  suitable,  the  formation  of  indol 
starts  almost  immediately.  Cole  and  Onslow*  have  de- 
scribed a  "  tryptic  broth,  "prepared  by  digesting  a  solution 
of  commercial  casein  with  trypsin,  as  being  eminently 
suitable  for  making  indol  tests.  It  contains  an  abundance 
of  tryptophane  and  other  amino-acids  in  the  free  state. 
The  growth  of  the  organisms  is  usually  very  luxuriant, 
and  the  element  of  uncertainty  about  the  appearance  of 
indol  is  abolished.  With  this  medium  the  production  of 
indol  by  a  typical  indol  former  can  be  detected  in  four  or 
five  hours. 

One  of  the  best  known  indol  formers  is  B.  coli,  an  almost 
universal  inhabitant  of  the  intestine.  Organisms  closely 
allied  to  it,  such  as  B.  typhosus  and  B.  paratyphosus  are 
unable  to  transform  tryptophane  into  indol  or  scatol. 

It  is  interesting  to  note  that  the  simultaneous  fermenta- 
tion of  glucose  inhibits  indol  production.  This  is  not  due 
to  the  fact  that  the  medium  becomes  acid  owing  to  the 
carbohydrate  fermentation.  It  indicates  that  the  meta- 
bolism and  enzyme  production  of  the  organisms  are 
profoundly  influenced  by  the  available  sources  of  energy. 
Since  a  large  number  of  diseases  are  caused  by  the  products 
of  bacterial  action,  a  complete  study  of  the  chemistry  of 
bacterial  growth  is  of  great  importance. 

For  the  following  experiments  the  appliances  and  technique  of  a  bacterio- 
logical laboratory  are  required. 

Preparation  of  "  tryptic  broti."  A  10  per  cent,  solution  of  commercial 
casein  is  prepared  as  described  in  Ex.  87,  A  (i.)  to  (vi.).  Toluol  is  added  as  an 
antiseptic,  but  the  sodium  fluoride  is  omitted.  Trypsin  is  then  added  and  the 

*  The  Lancet,  July  I,   1916,  p.  9. 


CH.  VIII.]  INDOL.  227 

digestion  carried  out  as  described  on  p.  88,  except  that  it  can  proceed  for  10  to 
1 6  days  if  convenient.  At  the  end  of  the  digestion  period  the  mixture  is 
transferred  to  a  flask,  treated  with  150  cc.  of  a  i  in  10  dilution  of  pure  concen- 
trated hydrochloric  acid,  heated  in  a  steamer  for  30  to  60  minutes  and  filtered. 
The  filtrate  is  treated  with  5  per  cent,  sodium  hydroxide  until  it  is  faintly 
alkaline  to  litmus.  The  resulting  fluid  or  "  stock  broth  "  can  be  preserved  in 
a  stoppered  bottle  for  a  very  considerable  period  if  a  little  toluol  be  added. 
One  part  of  the  stock  broth  is  treated  with  two  volumes  of  a  0-4  per  cent, 
solution  of  sodium  chloride  and  the  reaction  adjusted  to  PH  =  7-3  to  7-4  by  the 
method  given  in  Ex.  322,  Note  3.  The  "  tryptic  broth  "  thus  obtained  is 
distributed  into  convenient  flasks,  which  are  plugged  with  cotton  wool  and 
sterilised  in  the  autoclave. 

A  flask  (labelled  A)  of  the  cooled  sterile  broth  is  inoculated  with  a  culture 
of  B.  coli  (obtainable  from  a  bacteriological  laboratory)  under  the  precautions 
usually  adopted  to  prevent  accidental  contamination,  incubated  for  2  days  or 
longer.  For  comparison  an  uninoculated  flask  (labelled  B)  of  the  sterile 
broth  is  also  incubated. 

269.  Ehrlich's  test  for  indol.    To  about  3  cc.  of  A  and  of  B 

add  an  equal  volume  of  Ehrlich's  reagent  (seep.  390).  A  fine  red 
colour  appears  in  A,  but  not  in  B. 

NOTE. — If  the  test  does  not  succeed  it  is  usual  to  add  3  cc.  of  a  saturated 
solution  of  potassium  persulphate.  The  author  has  never  been  able  to 
recognise  the  advantage  of  this  in  improving  the  delicacy  of  the  test. 

270.  Nelson's  vanillin  test  for  indol.    To  about  3  cc.  of  A 
and  B  add  about  5  drops  of  a  5  per  cent,  solution  of  vanillin  in 
alcohol  and  then  about  3  cc.  of  pure  concentrated  hydrochloric  acid. 
An  orange  colour  is  produced  in  A,  due  to  the  presence  of  indol  or 
scatol.     In  B  a  slight  purplish  tint  may  develop  on  standing,  due  to 
a  reaction  with  unchanged  tryptophane. 

271.  Destruction  of  tryptophane  by  B.  coli.     To  5  cc.  of  A  and 

B  add  a  drop  or  two  of  acetic  acid  and  then  bromine  water,  drop  by 
drop,  until  no  further  increase  in  the  colour  is  obtained.  Warm 
slightly,  add  5  cc.  of  butyl  or  amyl  alcohol  to  each,  shake  and  allow 
to  stand.  The  alcohol  layer  is  more  deeply  coloured  in  B  than  in  A, 
owing  to  the  conversion  of  the  tryptophane  to  indol  and  scatol. 

272.  Tryptophane  is  the  only  mother-substance  of  the  indol 
bodies.     100  grams,  of  commercial  casein  are  hydrolysed  by  boiling 
with  500  cc.  of  i  in  4  sulphuric  acid  for  12  to  16  hours  under  a  reflux 
condenser.     The  resulting  dark  purple  solution  is  diluted  with  water 


228  COMPOSITION   OF   THE   DIGESTIVE   JUICES.  [CH.  VIII. 

and  the  sulphuric  acid  removed  by  the  addition  of  a  hot  saturated 
solution  of  baryta,  which  should  be  added  until  the  reaction  is  faintly 
alkaline  to  litmus  paper.  The  barium  sulphate  is  filtered  off  by  means 
of  a  Buchner  funnel.  The  filtrate  is  cautiously  treated  with  dilute 
sulphuric  acid  until  no  further  precipitate  is  obtained  and  filtered 
again.  The  nitrate  is  made  faintly  alkaline  to  litmus,  and  diluted, 
if  necessary,  to  make  a  total  volume  of  about  4  litres.  0-5  gram,  per 
cent,  of  sodium  chloride  is  added,  and  the  reaction  adjusted  to 
about  PH  =  7-35.  It  is  then  distributed  into  flasks  and  sterilised  in 
the  autoclave.  One  flask,  labelled  C,  is  inoculated  with  a  culture 
of  B.  coli  and  labelled  C.  Another  flask  is  treated  with  o-i  gram. 
per  cent,  of  pure  tryptophane,  heated  for  ten  minutes  on  a  boiling 
water  bath,  cooled,  inoculated  with  B.  coli,  and  labelled D.  Another 
flask,  labelled  E,  is  taken  as  a  control.  The  three  flasks  are  incu- 
bated for  2  to  5  days  at  38°  to  40°  C. 

(i.)  On  portions  of  the  three  fluids  perform  tests  for  indol  by 
Exs.  269  and  270.  They  are  only  obtained  in  D. 

(ii.)  Apply  the  glyoxylic  test  or  the  bromine  test  (Ex.  88, 
A  and  B)  for  tryptophane  to  E.  Neither  are  obtained,  owing  to  the 
destruction  of  tryptophane  during  the  acid  hydrolysis  of  the  casein. 
The  experiment  proves  that  tryptophane  is  the  only  amino-acid 
which  yields  indol  on  bacterial  decomposition. 


K.     Autolysis. 

When  an  organ  is  removed  from  the  body  and  incubated 
for  several  days  at  38°  C.  in  the  presence  of  an  antiseptic 
such  as  toluol,  it  is  found  that  a  certain  amount  of  the 
tissue  proteins  have  been  hydrolysed  to  amino-acids.  This 
disintegration  of  the  tissues  is  known  as  "autolysis."  It 
is  due  to  the  action  of  certain  proteolytic  enzymes  which 
seem  to  be  present  in  nearly  all  tissues,  and  which  are 
sometimes  known  as  "tissue  erepsins."  But  it  must  be 
noted  that  intestinal  erepsin  does  not  act  on  the  native 
proteins,  but  only  on  proteoses,  peptones  and  polypeptides. 
There  must  be  an  essential  difference  between  the  two 
enzymes. 


CH.  VIII.]  AUTOLYSIS.  22Q 

The  rate  and  degree  of  hydrolysis  varies  considerably 
with  the  nutritive  condition  of  the  tissue  at  the  moment 
of  death  and  on  the  reaction  of  the  medium  in  which  the 
autolysis  proceeds.  In  general  it  may  be  stated  that  it  is 
accelerated  by  the  previous  starvation  of  the  animal  and 
by  an  acid  reaction,  the  optimum  being  between  PH  =  5 
and  PH  =  6. 

"The  fact  that  autolysis  is  accelerated  by  starvation 
suggests  that  it  is  the  mechanism  by  means  of  which 
the  amino-acids  liberated  are  deaminised  to  ammonia  and 
an  acid.  The  latter  is  oxidised  to  CO2  and  water,  either 
directly  or  after  passing  through  the  stage  of  carbohydrate. 
These  products  being  removed  by  the  lungs,  the  net  result 
is  a  supply  of  ammonia  for  the  neutralisation  of  the  acid 
originally  causing  the  disturbance.  It  is  noteworthy  that 
the  products  of  autolysis  contain  much  more  ammonia 
than  is  found  in  the  products  of  action  of  trypsin  and 
erepsin. 

273.  The  autolysis  of  ox  kidney.  Ox  kidney  is  freed  from 
fat,  finely  minced  in  a  machine  and  weighed.  The  pulp  is  treated 
with  3  times  its  weight  of  0*2  per  cent,  acetic  acid  and  shaken  with 
toluol,  as  a  preservative.  The  mixture  is  incubated  for  3  or  4  days 
at  38°  C.  It  is  then  filtered  from  an  insoluble  residue  of  haematin, 
nuclein,  etc.  The  filtrate  is  treated  with  more  toluol  and  incubated 
for  another  7  to  9  days. 

A.  Boil  about  10  cc.    Note  the  heat  coagulation  of  undigested 
native  protein.     It  may  be  necessary  to  adjust  the  reaction  to  get 
complete  coagulation.    Filter  off  the  coagulum  and  to  a  portion  of 
the  filtrate  add  bromine  water,  drop  by  drop.     A  red  or  purple 
colour  indicates  the  presence  of  free  tryptophane  (see  Ex.  261). 

B.  Boil  another  portion  of  the  mixture  as  before.     Cool  10  cc. 
of  the  filtrate,  add  10  drops  of  strong  sulphuric  acid  and  cool  again. 
Add  an  equal  volume  of  the  mercuric  sulphate  reagent  of  Hopkins 
and  Cole  (see  p.  89),  mix  and  allow  to  stand  for  10  to  20  minutes.    A 
precipitate  is  obtained  which  consists  of  mercury  compounds  of 
tryptophane  and  of  the  purine  bases.     Filter. 


230 


COMPOSITION   OF  THE   DIGESTIVE   JUICES.  [CH.  VIII. 


Precipitate.  On 
small  portions  try 
the  glyoxylic  reac- 
tion for  tryptophane 
and  Millon's  reac- 
tion for  tyrosine. 
The  former  is  in- 
tense :  the  latter 
negative  or  feeble. 


Filtrate.  On  portions  apply  the  same 
two  reactions.  Millon's  is  intense :  the 
glyoxylic  is  negative  or  feeble.  To  the 
remainder  add  40  per  cent,  soda  drop  by 
drop  until  the  reaction  is  nearly  neutral. 
Filter. 


Precipitate.  Ap- 
ply Millon's  test.  A 
fairly  strong  reac- 
tion is  obtained. 


Filtrate.  Apply 
Millon's  test.  A 
negative  or  feeble 
reaction  is  obtained. 


Tryptophane  is  precipitated  by  the  mercuric  reagent,  even  in 
the  presence  of  7  per  cent,  sulphuric  acid.  Tyrosine  is  not  pre- 
cipitated in  this  strength  of  acid,  but  is  precipitated  in  a  solution 
that  is  only  faintly  acid. 

It  is  not  easy  to  obtain  crystals  of  tyrosine  or  leucine  by  direct 
evaporation  of  the  original  solution.  This  is  apparently  due  to  the 
presence  of  purine  bases  and  other  compounds.  If  these  be  first 
removed  by  precipitation  with  phosphotungstic  acid  in  3  per  cent, 
sulphuric  acid  solution  (the  fluid  also  being  acidified  to  this  extent) 
and  the  two  acids  be  removed  by  baryta,  the  filtrate  gives  crystals 
of  tyrosine  and  leucine  on  evaporation. 


L.     Oxidases,  Peroxidases,  and  Tyrosinase. 

The  mechanisms  concerned  in  the  oxidations  in  the 
body  of  such  relatively  stable  substances  as  fats  and 
carbohydrates  are  by  no  means  fully  understood.  It  is 
generally  believed  that  it  is  due  to  the  action  of  enzymes, 
which  are  either  very  unstable  or  are  only  active  when 
associated  with  living  protoplasm.  This  would  account 
for  the  fact  that  the  ingestion  of  protoplasmic  poisons,  such 
as  quinine,  has  a  distinct  inhibitor}7"  influence  on  various 
oxidative  processes  in  the  body.  There  is  an  increasing 
amount  of  evidence  to  indicate  that  the  sulphur  con- 


CH.  VIII.]  OXIDASES.  231 

stituents  of  the  cell  are  intimately  related  to  oxidative 
processes. 

Extracts  of  various  plant  tissues  possess  the  property 
of  inducing  the  oxidation  of  certain  aromatic  substances, 
either  directly  or  after  the  addition  of  hydrogen  peroxide. 
The  usual  nomenclature  is  that  an  oxidase  acts  directly, 
whilst  a  peroxidase  requires  the  addition  of  hydrogen 
peroxide.  One  view  as  to  the  mechanisms  concerned  is 
that  the  complete  oxidase  system  consists  of  a  peroxidase 
and  an  organic  peroxide.  The  peroxidase  deprives  the 
peroxide  of  an  atom  of  oxygen  and  transfers  it  to  the 
substance  that  is  oxidised  (the  acceptor).  This  view  can 
be  represented  as  follows  : 

(i.)     2  RO  +  02  =  2 

Atmospheric       Peroxide. 
Oxygen. 

/O 
(ii.)     R  "       |    +  Peroxidase   =  RO  +  Peroxidase  =  O 


_  ^___^ 

Oxidase  .  A  ctive 

Oxygen. 

(iii.)     Peroxidase  +  O  +  A     =      AO  +  peroxidase. 

Acceptor.    Oxidised 
product. 

On  this  view  the  expressed  plant  juices  or  extracts 
which  give  the  direct  oxidase  reaction  contain  an  organic 
substance  (RO)  which  can  form  a  peroxide.  When  this 
organic  substance  is  absent,  hydrogen  peroxide  must  be 
added  to  complete  the  oxidase  system. 

The  view  outlined  above  is  based  on  the  results  ob- 
tained when  the  acceptor  is  guiaconic  acid.  This  is  present 
in  tincture  of  guiacum  resin  and  is  converted  to  guiacum 


232  COMPOSITION   OF   THE   DIGESTIVE   JUICES.  [CH.  VIII. 

blue  by  oxidation.  Various  other  aromatic  substances, 
such  as  benzidine,  a-naphthol  and  para-phenylene-diamine 
can  be  used  as  acceptors,  but  in  such  cases  it  is  found  that 
hydrogen  peroxide  must  usually  be  added,  even  to  an 
extract  which  behaves  as  a  complete  oxidase  system  to- 
wards guiacum.  Some  very  interesting  results  have 
recently  been  obtained  by  Mrs.  Muriel  Wheldale  Onslow, 
relating  the  establishment  of  the  oxidase  system  in  certain 
plants  to  the  browning  that  takes  place  on  injury  and  to 
the  preliminary  oxidation  of  some  aromatic  cell  constituent. 
They  will  shortly  be  published  in  the  Biochemical  Journal. 

The  tyrosinases  are  oxidising  enzymes  which  act  on 
tyrosine.  They  also  act  on  substances  of  allied  constitu- 
tion, such  as  ^-cresol.  The  relationships  of  these  plant 
enzymes  to  the  mechanism  of  the  oxidations  occurring  in 
the  animal  body  is  not  at  present  certain. 

Preparation  of  guiacum  tincture.  Take  the  inner  portions  of  a  lump  of 
resin  and  make  a  i  -5  per  cent,  solution  in  95  per  cent,  alcohol,  by  heating  in  a 
flask  in  a  boiling  water  bath.  Add  some  adsorbent  charcoal,  boil  for  about  5 
minutes  and  filter.  The  solution  should  be  freshly  prepared. 

A  more  reliable  preparation  of  guiaconic  acid  is  described  by  Lyle  and 
Curtman  (Journ.  Biol.  Chem.,  xxxiii.,  p.  i). 

274.  Potato  oxidase.    Thoroughly  wash  and  scrub  a  potato. 
Peel  it  and  pound  the  peel  in  a  mortar  with  a  little  cold  water. 
Filter.    Treat  a  small  amount  of  the  nitrate  with  a  few  drops  of  the 
tincture  of  guiacum.    A  blue  colour  is  obtained  after  a  short  time. 
Boil  a  few  cc.  of  the  aqueous  extract,  cool  and  add  guiacum.    No 
blue  colour  is  obtained,  showing  that  the  enzyme  is  destroyed  by 
boiling. 

275.  Conversion  of  Potato  oxidase  to  a  per  oxidase.     Heat 
about  5  cc.  of  the  potato  extract  to  65°  C.  for  10  minutes  in  a  water 
bath.    Divide  the  solution  into  two  portions,  A  and  B.    To  A  add  a 
few  drops  of  guiacum  tincture  and  a  few  drops  of  hydrogen  peroxide. 
To  B  add  a  few  drops  of  guiacum  tincture.    A  generally  goes  blue, 
whilst  B  generally  gives  a  negative  or  very  feeble  reaction. 

NOTE. — The  organic  peroxide  is  more  unstable  to  heat  than  the  peroxi- 
dase.  After  heating,  the  solution  requires  the  addition  of  hydrogen 
peroxide. 


CH.  VIII.]  TYROSINASE.  233 

276.  Horseradish   peroxidase.      Horseradish    scrapings    are 
soaked  in  alcohol,  filtered  and  dried  in  a  thin  layer.    The  dried 
scrapings  are  extracted  with  water  and  filtered.     Place  2  or  3  cc. 
in  labelled  test-tubes  and  try  the  following  experiments,  a  few  drops 
of  guiacum  tincture  or  of  10  volumes  hydrogen  peroxide  where 
indicated. 

A.  Extract  4-  guiacum. 

B.  Extract  +  guiacum  +  H2O2. 

C.  Guiacum  +  H2O2. 

D.  Boiled  extract  +  guiacum  4-  H2O2. 

If  satisfactory  reagents  are  employed  a  blue  colour  is  only 
produced  in  B. 

277.  Potato  tyrosinase.    Prepare  an  extract  of  potato  peel 
as  described  in  Ex.  274.     Boil  a  little  tyrosine  with  about  5  cc.  of 
distilled  water  and  cool  to  about  40°  C.     Make  the  following  mix- 
tures : — 

A.  Extract  +  tyrosine  suspension. 

B.  Extract  alone. 

C.  Boiled  extract  +  tyrosine  suspension. 

Incubate  at  37°  C.  and  note  the  appearance  at  intervals.   Both 
A  and  B  darken,  but  A  much  more  than  B. 


CHAPTER   IX. 
THE   COAGULATION   OF   BLOOD. 

In  spite  of  the  immense  amount  of  work  that  has  been 
done  on  the  subject,  it  is  impossible  to  explain  fully  the 
fact  that  when  blood  is  shed  it  rapidly  sets  to  a  jelly,  which 
subsequently  contracts.  The  clot  consists  of  the  corpuscles 
entangled  in  a  contracting  meshwork  of  fibrin  :  the  yellowish 
fluid  that  exudes  is  the  serum. 

The  following  account  of  some  of  the  factors  concerned 
and  the  appended  scheme  of  their  interaction  does  not 
allow  for  a  great  deal  of  important  work  that  has  been  done 
in  recent  years.  It  is  almost  certain  that  the  phenomenon 
is  considerably  more  complicated  than  that  indicated 
below. 

Factors  concerned. 

1.  Fibrinogen,  a  globulin,  present  in  blood-plasma. 
It  is  soluble  in  dilute  salt  solutions,  acids  and  alkalies, 
insoluble  in  distilled  water.     It  coagulates  at  57°  C.     It  is 
precipitated   by  half-saturation  with  sodium  chloride. 

2.  Pro-thrombin,  a  substance  of  unknown  composi- 
tion, found  in  plasma,  attached  to  the  fibrinogen.     It  is 
destroyed  by  boiling. 

3.  Thrombokinase,   a  substance  found  in  all  tissues 
and  also  liberated  in  the  blood  by  the  disintegration  of 
leucocytes  and  blood-platelets.     It  converts  pro-thrombi n 
into  thrombin,  under  certain  conditions. 

4.  Calcium  salts,  found  in  plasma,  and  necessary  for 
the  action  of  thrombokinase.     The  calcium  salts  must  be 
of  such  a  nature  that  they  are  ionised  in  solution. 


CH.  IX.]  THE   COAGULATION   OF   BLOOD.  235 

5.  Thrombin,  a  ferment  formed  by  the  interaction 
of  2,  3  and  4.  It  probably  splits  fibrinogen  into  serum- 
globulin  and  fibrin.  The  latter,  being  insoluble  in  the 
constituents  of  normal  plasma,  comes  out  of  solution  and 
with  the  corpuscles  forms  the  clot. 

The  scheme  on  the  following  page  represents  the  inter- 
action of  the  above  factors. 
Coagulation  is  hindered  by 

1.  Cooling. 

2.  Substances    which    precipitate    calcium    salts,    or 
convert   the   calcium   into   the   non-ionised   condition,   as 
oxalates,  citrates  and  soap  solutions. 

3.  Alkalies,  which  prevent  the  liberation  of  throm- 
bokinase  by  the  corpuscles,  delay  the  action  of  thrombin, 
and  tend  to  dissolve  fibrin. 

4.  Strong  salt  solutions,  which  have  a  similar  action. 

5.  Anti-thrombin,  a  substance  found  in  small  amounts 
in  the  plasma,  and  in  relatively  large  amounts  in  extracts 
of  the  head  of  the  leech.     It  combines  with  thrombin  to 
render  it  inactive. 

6.  Anti-kinase,   found  in  the  blood,   after  the  slow 
injection  into  the  blood  stream  of  certain  substances,  as 
tissue-extracts,   certain  snake-venoms,   etc. 

7.  Fluorides,  which  precipitate  calcium  salts  and  pre- 
vent the  liberation  of  thrombokinase. 

Preparation  of  fibrin  ferment  (thrombin).  Blood  serum  is  treated  with 
four  or  five  times  its  volume  of  strong  alcohol,  well  stirred  and  allowed  to 
stand  for  two  or  three  days.  The  precipitate  is  collected,  dried  on  filter  paper 
in  the  air,  and  extracted  with  water.  The  filtered  extract  contains  fibrin 
ferment. 

Preparation  of  "salted"  plasma.  Two  litres  of  water  are  placed  in  a 
large  bottle  or  jar  (provided  with  a  tightly-fitting  stopper)  and  the  level  of 
the  fluid  marked  by  a  label.  The  water  is  poured  off  and  400  cc.  of  a  saturated 
solution  of  magnesium  sulphate  substituted.  Blood  is  collected  in  the  bottle 
till  the  level  is  reached,  care  being  taken  to  ensure  thorough  mixing  with  the 
salt  solution  by  stopping  the  flow  of  blood  from  time  to  time  and  turning  the 
bottle  upside  down.  The  corpuscles  are  removed  by  centrifugalisation  and 
the  plasma  pipetted  off.  It  should  be  kept  in  a  refrigerator  till  required. 

278.  The  clotting  of  salted  plasma.  Take  2  cc.  of  salted 
plasma  in  a  test-tube,  add  10  cc.  of  water,  and  divide  into  two 
portions,  A  and  B.  To  A  add  a  few  drops  of  fibrin  ferment  (or  of 


236 


THE  COAGULATION  OF  BLOOD. 


[CH    IX, 


CH.  IX.]  THE   COAGULATION  OF   BLOOD.  237 

serum).  Place  both  tubes  in  the  warm  bath  at  40°  C.  and  examine 
from  time  to  time.  Clotting  takes  place  in  both  tubes,  but  much 
more  rapidly  in  A  than  in  B. 

NOTE. — Dilution  with  water  decreases  the  concentration  of  the  magnesium 
sulphate,  so  that  any  fibrin  formed  by  the  ferment  (which  can  now  act  on  the 
fibrinogen)  becomes  insoluble  in  this  low  concentration  of  salt. 

279.  The  preparation  of  fibrinogen.     To  20  cc.  of  the  salted 
plasma  add  an  equal  volume  of  a  saturated  solution  of  sodium 
chloride.     A  precipitate  of  fibrinogen  is  formed.     Allow  the  tube  to 
stand  for  a  few  minutes  and  then  filter  through  a  small  paper. 
Scrape  the  precipitate  off  the  paper  and  treat  it  with  about  5  cc.  of 
5  per  cent,  sodium  chloride.     The  fibrinogen  dissolves. 

NOTE. — If  bird's  blood  be  drawn  directly  into  a  clean  vessel  in  such  a  way 
that  contact  with  the  tissues  is  absolutely  avoided,  it  clots  very  slowly.  This  is 
because  the  leucocytes  are  very  stable  and  do  not  liberate  thrombokinase.  If 
this  blood  be  centrifugalised  at  once,  a  non-clotting  plasma  is  obtained. 
Fibrinogen  can  readily  be  prepared  from  this  by  the  method  given  in  Ex.  33. 
The  suspension  so  obtained  is  dissolved  in  dilute  salt  solution. 

280.  Divide  the  solution  thus  obtained  into  two  portions,  C 
and  D.     To  C  add  two  drops  of  fibrin  ferment.     Place  both  tubes 
in  the  warm  bath  and  observe  them  at  intervals.     C  clots  rapidly  ; 
D  very  slowly. 

Preparation  of  oxalate  plasma.  Blood  is  drawn  as  in  the  prepara- 
tion of  salted  plasma  into  a  bottle  which  has  200  cc.  of  a  i  per  cent,  solution 
of  potassium  oxalate  in  place  of  the  400  cc.  of  saturated  magnesium  sulphate. 
The  plasma  is  separated,  as  before,  by  centrifugalisation. 

281.  The  clotting  of  oxalate  plasma.     Dilute  5  cc.  of  the 
plasma  with  10  cc.  of  distilled  water  and  divide  into  three  portions, 
E,  F,  and  G.    To  E  add  a  few  drops  of  i  per  cent,  calcium  chloride ; 
to  F  a  few  drops  of  fibrin  ferment  or  serum.      Place  the  three  tubes 
on  the  water  bath  and  observe  them  at  intervals.     E  clots  in  a  few 
minutes ;  F  clots  slowly ;  G  does  not  clot. 

Preparation  of  fluoride  plasma.  This  is  prepared  as  oxalate  plasma, 
using  a  3  per  cent,  solution  of  sodium  fluoride  in  place  of  the  i  per  cent, 
potassium  oxalate. 

282.  The  clotting  o!  fluoride  plasma.     Dilute  5  cc.  with  10 
cc.  of  water  and  divide  into  three  portions/H,  K,  and  L.     To  H  add 
a  few  drops  of  i  per  cent,  calcium  chloride ;  to  K  a  few  drops  of  fibrin 
ferment.     Place  the  three  tubes  in  the  warm  bath  and  observe  them 
at  intervals.     K  clots  rapidly ;  H  and  L  do  not  clot. 


CHAPTER   X. 

THE    RED    BLOOD    CORPUSCLES    AND 
THE    BLOOD    PIGMENTS. 

A.     The  Laking  of  Blood. 

The  red  corpuscles  consist  of  an  envelope  and  meshwork 
called  the  stroma,  which  encloses  a  solution  of  haemoglobin 
and  various  salts.  The  stroma  consists  of  a  protein, 
probably  a  histone,  with  which  is  associated  a  lipoid 
material,  related  to  cholesterin  and  lecithin.  The  envelope 
behaves  as  a  semi-permeable  membrane  to  a  great  many 
solutions,  readily  allowing  water  to  pass  into  or  from  the 
corpuscle,  but  preventing  the  passage  of  most  salts  and 
other  dissolved  substances.  Thus,  if  the  corpuscles  are 
placed  in  a  solution  which  has  a  higher  osmotic  pressure 
than  the  fluid  within  the  corpuscles,  water  passes  out  of  the 
corpuscle,  which  therefore  shrinks.  Such  fluids  are  called 
"  hypertonic."  If  they  be  placed  in  fluids  of  a  lower 
osmotic  pressure  ("hypotonic"),  water  passes  into  the 
corpuscle  to  equalise  the  pressures,  but  salts  cannot  pass 
out.  The  corpuscles  swell  and  the  expansion  may  be 
sufficient  to  lead  to  the  disruption  of  the  envelope,  so  that 
the  enclosed  haemoglobin  passes  into  the  body  of  the 
solution.  This  bursting  of  the  corpuscles  is  known  as 
taking  or  haemolysis.  A  solution  of  the  same  osmotic 
pressure  as  that  of  the  fluid  within  the  corpuscle  is  said 
to  be  "isotonic"  or  "normal."  For  mammalian  blood 
0-9  per  cent,  sodium  chloride  is  normal ;  for  frog's  blood, 
0-65  per  cent.  Other  physical  means  of  inducing  hae- 
molysis are  by  repeatedly  freezing  and  thawing  the  blood, 
or  by  warming  to  60°  C. 

The  envelopes  can  also  be  ruptured  by  chemical  means. 
Certain  substances,  such  as  the  bile  salts,  ether,  chloroform, 


CH.  X.]  HAEMOLYSIS.  239 

acids,  alkalies,  soaps  and  "  saponins  "  cause  the  blood  to  be 
laked.  Some  of  these  act  by  dissolving  the  lipoids  of  the 
envelope  and  stroma  ;  others  possibly  act  on  the  proteins. 

If  the  washed  corpuscles  be  suspended  in  normal 
saline  containing  various  buffer  solutions,  it  will  be  found 
that  haemolysis  takes  place  in  all  solutions  acid  to  PH=S*I. 

Lecithin  and  cholesterol  seem  to  be  somewhat  antagon- 
istic in  respect  to  haemolysis,  the  former  accelerating  and 
the  latter  inhibiting  the  phenomenon. 

Certain  pathogenic  organisms  produce  specific  hae- 
molysins,  notably  the  tetanus  bacillus.  The  poisonous 
action  of  some  snake  venoms  is  in  part  due  to  the  rapid 
destruction  of  the  red  corpuscles. 

Haemolysis  is  brought  about  by  the  absorption  into 
the  system  of  a  large  number  of  chemical  substances,  e.g. 
arsine,  pyrogallol,  toluylenediamine. 

Another  method  of  inducing  haemolysis  is  by  the 
addition  of  certain  organic  substances  developed  in  certain 
animals.  Thus  rabbit's  corpuscles  that  have  been  washed 
with  isotonic  saline  are  laked  when  treated  with  the 
blood  serum  of  a  dog.  This  haemolytic  power  of  dog's 
serum  on  rabbit's  blood  is  very  much  increased  by  previously 
injecting  the  dog  with  rabbit's  blood'. 

283.  Have  two  burettes,  one  containing  i  per  cent,  sodium 
chloride,  the  other  distilled  water. 

Label  a  series  of  clean,  dry  test-tubes  A,  B,  C,  etc. 

In  A  place  4-5  cc.  NaCl  and  5-5  cc.  H2O  -  -45  %  NaCl. 
"     »      5  >»  5  »    ~  "5    » 

C      „      5*5  „  4'5          „    =  -55  „ 

D     „      6  „  4  „    =  -6    „ 

E     „      6-5  „  3-5          „    =  -65  „ 

F      „      7  ..  3  „     =  7    » 

To  each  tube  add  three  drops   of  fresh  defribinated  blood, 


240  THE   RED   BLOOD   CORPUSCLES.  [CTI.  X. 

mix  by  inverting  and  allow  the  tubes  to  stand  for  a  few  minutes. 
A  will  be  translucent,  the  corpuscles  being  fully  laked.  F  will  be 
opaque.  Note  the  dilution  which  just  causes  laking.  It  is  generally 
about  0-55  per  cent. 

NOTE. — The  solution  that  just  causes  laking  is  hypotonic  to  the  blood, 
indicating  that  the  corpuscles  can  absorb  a  considerable  quantity  of  fluid  before 
the  envelope  is  ruptured. 

284.  To  5  cc.  of  0-9  per  cent,  sodium  chloride  add  some  ether 
and  shake  vigorously.     Then  add  three  drops  of  blood,  mix  by 
inversion.      Warm  gently  and  add   a   few  more    drops   of   ether. 
The  blood  is  laked. 

NOTE. — It  is  essential  that  pure  ether  be  used.  Should  the  ether  be 
contaminated  with  acid  the  blood  is  precipitated  and  the  pigment  converted 
to  acid  haematin. 

285.  To  a  0-2  per  cent,  solution  of  bile  salts  in  normal  saline 
add  three  drops  of  blood.     Mix  and  warm  to  37°  C.     The  blood  is 
generally  laked,  though  the  experiment  does  not  always  succeed. 

286.  Add  some  blood  to  a  2  per  cent,  solution  of  urea  in 
water.     The  blood  is  laked. 

287.  Repeat  the  experiment  with  a  2  per  cent,  solution  of 
urea  in  normal  saline.     The  blood  is  not  laked. 

NOTE. — A  solution  of  urea  behaves  like  water  as  regards  the  corpuscles. 
No  matter  what  concentration  is  used  the  corpuscles  take  up  water  from  the 
surrounding  fluid.  In  other  words,  the  envelope  of  the  corpuscle  does  not  act 
as  a  semipermeable  membrane  as  regards  urea. 

B.    Haemoglobin  and  its  Derivatives. 

Haemoglobin  (Hb)  is  a  compound  protein,  being  a 
member  of  the  group  of  chromoproteins.  It  is  formed  by 
the  union  of  a  pigmented  non-protein  substance  containing 
iron,  and  called  haematin  (Hn),  with  globin,  a  member 
of  the  histone  group  of  proteins. 

It  is  soluble  in  water  and  dilute  salt  solutions  :  in- 
soluble in  ether  and  alcohol. 

It  is  decomposed  b}^  acids  and  alkalies  into  haematin 
and  globin.  It  is  decomposed  and  coagulated  by  heat. 


CH.  X.]  OXYHAEMOGLOBIN.  24! 

It  forms  compounds  with  oxygen  and  carbon  mon- 
oxide, called  oxyhaemoglobin  (Hb-O2)  and  carboxyhae- 
moglobin  (Hb-CO).  Both  are  dissociated  into  Hb  and  the 
gas  by  exposure  to  a  vacuum.  Hb-CO  is  much  more 
stable  than  Hb-O2,  and  the  avidity  of  Hb  for  CO  is  more 
than  130  times  greater  than  the  avidity  of  Hb  for  O2.  A 
small  percentage  of  CO  in  the  air  breathed  will  thus  result 
in  the  formation  of  relatively  considerable  amounts  of 
Hb-CO  in  the  blood.  This  can  be  converted  into  Hb-O2 
by  exposure  to  a  high  tension  of  O2,  such  as  is  obtained  by 
breathing  pure  O2. 

The  Hb-O2  obtained  from  certain  animals  crystallises 
readily,  but  the  crystals  differ  somewhat,  according  to  the 
animal  from  which  they  are  obtained.  Also  the  volume 
of  O2  combining  with  i  gram,  of  Hb  varies,  the  figure  for 
the  horse  being  i  -34  cc.  of  O2  per  gram,  of  Hb.  The  oxygen 
is  probably  united  to  the  iron  of  the  haematin  molecule, 
the  reaction  Fe  +  O2  <  >  FeO8  being  the  basis  of  the 
reaction  Hb  +  O 


^u         ,.     volume  of  O2  evolved  in  cc.  .        „    -,   ,, 
The  ratio  -  -  is  called  the 

weight  of  iron  in  grams. 

specific  oxygen  capacity. 


Theoretically  it  is 

O2  _  i  molecular  volume  O2  _  22,394 
Fe       i    gram,    molecule     Fe       55*85 


401 


Recent  analyses  of  the  blood  of  various  animals  have 
given  the  value  401-8  which  agrees  very  closely  with  the 
theoretical. 

The  volume  of  oxygen  loosely  held  by  i  gram,  of 
Hb-O2  is  i  »345  cc. 

So    the    minimum    molecular    weight    of   ox3^haemo- 


globin  is  -  =  16,712. 

1-345 


CALIFORNIA  COLLEfli 

of   PHARMACf  . 


242 


THE   RED   BLOOD   CORPUSCLES. 


[CH.  X 


The  method  of  formation  of  certain  of  the  derivatives 
of  haemoglobin  can  be  represented  as  follows  :— 


METHAEMOGLOB1N 


.s'/ 

1 

1 

§ 

04                                          S 

«                      -a 

Globin 

.44 

§ 

«                    jS 

-f~ 

. 

ACID               Dilute 
HAEMATIN   ^  Acids 

B 

OXYHAEMOGLOBIN 

"U 

i 

1         1 

co 

*                                T3 

| 

0                        ^ 

X 

f 

Dilute  ,  ALKALINE 

Alkalies     HAEMATIN  +  Globin 


HAEMOGLOBIN  ^^^  HAEMOCHROMOGEN 


Jron+    ACID    HAEMATOPORPHYR1N 


ALKALINE    HAEMATOPORPHYRIN 

289.    Crystallisation  of  oxyhaemoglobin  (Rapid  method).    To 

a  few  cc.  of  defibrinated  dog's  blood  in  a  test-tube  add  ether,  drop 
by  drop,  till  the  blood  is  completely  laked.  Add  to  the  blood  a 
pinch  of  finely  powdered  ammonium  oxalate ;  allow  the  salt  to  dis- 
solve by  gentle  shaking,  and  let  the  tube  stand.  Crystals  of  oxy- 
haemoglobin separate  out,  especially  if  the  solution  is  cooled  to 
o°  C.  by  means  of  ice.  Examine  them  microscopically,  and  note 
that  they  are  in  the  form  of  thin  rhombic  prisms. 

Make  a  drawing  of  the  crystals. 


NOTE. — This  experiment  does  not  always  succeed  as  described.  If  the 
blood  fails  to  crystallise  out  in  an  hour,  place  a  drop  on  a  slide,  spread  it  out  to 
form  a  thin  layer  and  leave  it  for  five  minutes ;  cover  with  a  slip  and  note  the 
crystals  of  oxyhaemoglobin  that  form  at  the  edges. 


CH.  X.] 


THE   SPECTROSCOPE. 


243 


B 


C.     The  Spectroscopic  Examinations  of  the  Blood  Pigments. 

The  use  of  the  Direct-vision  Spectroscope. 

The  instrument  described  is  the  small  pocket  spectroscope,  with  wave- 
length scale  attached,  manufactured  by  Zeiss  and  Co.  It  is  to  be  hoped  that 
an  equally  good  instrument  of  home  manufacture 
will  soon  be  forthcoming.  The  instrument  (fig.  30) 
consists  of  two  tubes.  The  shorter  tube  A  con- 
tains a  transparent  photographic  scale  of  wave- 
lengths, with  a  mirror  to  project  its  image  into  the 
field  of  vision.  By  means  of  the  tube  D  this  scale 
can  be  focussed,  and  by  the  screw  F  it  can  be 
adjusted  to  its  proper  position.  The  tube  G  con- 
tains a  series  of  alternating  prisms  of  crown  and 
flint  glass,  arranged  to  allow  the  spectrum  to  be 
observed  by  the  eye  in  the  line  of  the  tube.  The 
tube  B  which  slides  on  G  has  a  vertical  slit,  the 
width  of  which  can  be  adjusted  by  turning  the 
collar  E. 

To  adjust  the  spectroscope:  see  that  D  and  B 
are  pushed  in  as  far  as  they  will  go.  Look  through 
C  towards  the  light  with  A  to  your  left.  Cautiously 
turn  E  till  the  spectrum  is  just  visible.  (It  is  most 
important  to  use  an  extremely  narrow  slit.)  Slide 
B  out  very  slowly  (in  most  instruments  for  3^ 
divisions  as  marked  on  the  barrel  G)  till  fine  black 
vertical  lines  can  be  seen  in  the  spectrum,  and 
notice  particularly  a  fine  black  line  immediately  to 
the  left  of  the  narrow  strip  of  yellow.  This  line  is 
known  as  the  D  line  of  Fraunhofer.  The  wave- 


C 


Fig.  30. — Zeiss'  direct- 
vision  spectroscope  with 
wave-length  scale  (x£). 


length  of  it  is  -59  p,  a  position  indicated  on  the  scale  by  the  division  marking  it 
(the  one  to  the  right  of  0-6)  being  produced  further  down  than  any  other.  If 
necessary  alter  the  position  of  the  scale  by  turning  the  screw  F  until  the  D  line 
exactly  coincides  with  the  division  mentioned.  If  the  instrument  has  to  be 
adjusted  at  night-time,  when  the  D  line  cannot  be  observed,  set  the  scale 
by  use  of  the  emission-spectrum  of  sodium  (obtained  by  placing  a  few  crystals 
of  common  salt  on  the  wick  of  a  spirit  lamp).  The  emission  spectrum  of 
sodium  exactly  corresponds  to  the  D  line.  The  scale  is  so  drawn  that,  if  it  be 
set  in  position  as  described,  the  wave-length  of  light  in  any  part  of  the  visible 
spectrum  can  be  read  directly. 

The  numbers  on  the  scale  indicate  wave-lengths  in  thousandths  of  a 
millimetre,  the  unit  being  i  p..  In  the  more  recent  patterns  the  wave-lengths 
are  given  in  millionths  of  a  millimetre,  the  unit  being  i  \.  Thus  the  wave 
length  of  the  D  line  is  589  X.  The  other  Fraunhofer  lines  that  can  be  readily 
observed  with  the  instrument  are  C  (657  X),  E  (527  X),  b  (518  X)  and  F(486X)« 

To  observe  absorption  spectra :  slightly  open  the  slit  of  the  spectroscope* 
thus  obtaining  a  better  illumination.  Direct  the  instrument  to  the  light,  and 
place  the  test-tube  containing  the  fluid  to  be  examined  directly  in  front  of, 
and  touching,  the  tube  B,  with  its  axis  parallel  to  the  slit,  taking  care  not  to 
interfere  wth  the  illumination  of  the  scale.  With  strong  solutions  of  certain 
pigments  observed  in  this  way  it  is  often  difficult  to  avoid  illuminating  the  two 
ends  of  the  spectrum,  the  light  being  reflected  from  the  sides  of  the  tubes,  and 
not  passing  through  the  solution.  To  obviate  this  it  is  perhaps  better  to  place 
the  solution  in  a  beaker,  remembering  that  the  absorption  of  light  increases 


244  THE  RED  BLOOD  CORPUSCLES.  [CH.  X. 

with  the  depth  of  layer  examined,  as  well  as  with  the  concentration  of  the  pig- 
ment. For  accurate  work  the  haematoscope  should  be  employed.  This  is  a 
vessel  with  parallel  glass  slides  i  cm.  apart. 

In  handling  the  instrument  the  screw  F  is  very  liable  to  be  turned,  and 
so  the  position  of  the  scale  to  be  shifted.  From  time  to  time,  therefore,  the 
slit  should  be  narrowed,  and  an  observation  made  to  ascertain  whether  any 
shifting  of  the  scale  in  reference  to  the  D  line  has  occurred. 

Record  the  absorption  of  light  of  the  various  pigment  solutions  on  the 
blank  scale,  to  be  found  on  page  372.  Fill  in  with  black  pencil  marks  the  exact 
parts  of  the  spectrum  where  light  is  absorbed,  leaving  the  remainder  blank. 
It  will  not  be  found  advisable  to  use  coloured  pencils. 

290.  Oxyhaemoglobin.    Take  5  cc.  of  distilled  water  in  a  test- 
tube,   and  add  one  drop  of  defibrinated  blood,  shake  well  and 
observe  the  spectrum  of  dilute  oxy haemoglobin.      There  are  two 
absorption  bands  in  the  green.     The  one  near  the  D  line  (the  a  band) 
is  somewhat  narrower  and  darker  than  the  ft  band.     The  middle  of 
a  is  about  X  578,  and  that  of  ft  about  X  540. 

291.  Add  two  more  drops  of  defibrinated  blood  and  examine 
again.     The  spectrum  has  become  very  much  cut  off,  especially  at 
the  blue  end  :  the  absorption  bands  have  probably  merged  into  one, 
leaving  a  little  patch  of  blue  light  and  a  broader  belt  of  red  light  on 
the  two  sides.     If  this  effect  has  not  been  produced,  add  a  little 
more  blood  or  dilute  with  water.     Record  the  spectrum  of  the 
solution  on  the  chart  as  that  of  a  medium  solution  of  oxyhaemo- 
globin. 

292.  Add  another  drop  or  two  of  defibrinated  blood,  and  note 
that  the  blue  light  becomes  absorbed,  light  only  coming  through  in 
the  red.     (Strong  solution.)     If  the  concentration  is  still  further 
increased,  the  red  also  is  absorbed. 

NOTE. — It  is  important  to  observe  that  a  medium  solution  of  oxyhaemo- 
globin  has  a  single  band  in  the  green. 

293.  Haemoglobin  (reduced   haemoglobin).     Treat  5  cc.  of 
water  with  one  drop  of  defibrinated  blood  and  thus  obtain  a  solution 
of  oxyhaemoglobin  of  such  a  strength  that  two  well-marked  absorp- 
tion bands  can  be  observed.     Add  two  drops  of  a  solution  of  am- 
monium sulphide,  mix  and  warm  to  about  50°  C.,  avoiding  any 
unnecessary  shaking :  or  if  Stokes'  fluid  is  obtainable,  add  two  or 
three  drops,  in  which  case  there  is  no  necessity  to  warm.     Note,  in 


CH.  X.]  CARBOXYHAEMOGLOBIN.  245 

the  latter  case,  that  the  bright  scarlet  colour  of  oxyhaemoglobin 
gives  place  to  the  less  vivid  colour  of  reduced  haemoglobin.  Ex- 
amine the  solution  spectroscopically.  There  is  a  single  broad 
band  in  the  green  which  overlaps  the  space  enclosed  by  the  two 
bands  of  oxyhaemoglobin,  and  is  fainter  than  either.  Its  centre  is 
about  X  565. 

NOTE. — Stokes'  fluid  is  prepared  as  follows  :  dissolve  3  grams,  of  ferrous 
sulphate  in  cold  water  :  add  a  cold  aqueous  solution  of  2  grams,  of  tartaric  acid 
and  make  the  solution  up  to  100  cc.  with  water.  Immediately  before  use  add 
strong  ammonia  until  the  precipitate  first  produced  is  redissolved.  It  rapidly 
absorbs  atmospheric  oxygen  and  must,  therefore,  be  freshly  prepared.  Its 
great  advantage  over  ammonium  sulphide  is  that  it  can  be  used  in  the  cold, 
whilst  with  the  sulphide  the  solution  must  be  warmed. 

294.  Place  your  thumb  over  the  top  of  the  test-tube  contain- 
ing  the   reduced   haemoglobin   and   shake   vigorously.     Examine 
immediately  with  the  spectroscope,  and  note  that  the  two  bands  of 
oxyhaemoglobin  have  reappeared  owing  to  the  oxidation  of  the 
haemoglobin  by  the  oxygen  of  the  air.     If  the  tube  be  allowed  to 
stand  for  a  short  while,  reduction  may  occur  again  from  excess  of 
reducing  reagent  present. 

295.  Carboxyhaemoglobin.      Obtain    some    CO-haemoglobin 
that  has  been  prepared  by  passing  a  stream  of  carbon  monoxide  or 
coal-gas  through  a  solution  of  oxyhaemoglobin.     Notice  the  peculiar 
bluish  tinge  of  the  solution.     Examine  a  portion  spectroscopically, 
and,  if  necessary,  add  water  till  two  well-marked  bands  are  visible. 
Note  that  they  are  very  similar  to  the  two  bands  of  oxyhaemoglobin. 
Accurate   observation,   however,   will   show   that   they   are  both 
slightly  nearer  the  violet  end  of  the  spectrum,  the  middle  of  a 
being  X  572  and  of  ft  X  535. 

NOTES. — i.  A  small  amount  of  caprylic  alcohol  added  to  the  blood 
facilitates  the  preparation  of  Hb-CO  in  preventing  excessive  frothing. 

2.  If  the  student  can  satisfy  himself  of  the  difference  between  the  position 
of  the  absorption  bands  of  Hb-O2  and  Hb-CO,  he  can  always  obtain  a  sample 
of  Hb-O2  for  comparison  with  an  unknown  solution  by  pricking  his  finger. 

296.  Take  a  portion  of  the  diluted  solution  of  CO-haemoglobin 
just  examined,  treat  it  with  a  few  drops  of  ammonium  sulphide, 
warm  in  a  bath  at  50°  C.  for  three  minutes  and  examine  with  the 
spectroscope.     No  change  takes  place  in  the  spectrum.     (Distinction 
from  oxyhaemoglobin.) 


246  THE  RED  BLOOD  CORPUSCLES.  [CH.  X. 

297.  In  two  test-tubes  place  2  or  3  cc.  of  solutions  of  oxy- 
haemoglobin  and  CO-haemoglobin  of  the  same  depth  of  colour. 
Fill  the  test-tubes  with  water  and  mix  well.     Note  that  the  CO- 
haemoglobin   takes   on   a  well-marked   carmine   tint,   whilst   the 
oxyhaemoglobin  turns  }^ellow.     This  simple  test,  which  can  only  be 
seen  on  extreme  dilution,  rapidly  serves  to  distinguish  the  two 
compounds. 

298.  Methaemoglobin.      To   5  cc.    of  water  add  four  drops 
of  defibrinated  blood.      To  the  strong  solution  of  oxyhaemoglobin 
thus  formed  add  two  drops  of  a  saturated  solution  of  potassium 
ferricyanide.     The  colour  of  the  solution  changes  to  a  chocolate- 
brown.     Examine  with  the  spectroscope :  there  is  visible  a  promi- 
nent band  in  the  red,  with  its  centre  at  about  A  630.     There  is 
marked  absorption  of  the  blue  end  of  the  spectrum.     Dilute  with 
an  equal  bulk  of  water  and  examine  again  :  two  faint  bands  appear 
in  the  green  in  the  position  of  the  bands  of  oxyhaemoglobin. 

299.  Dilute  the  solution  of  met  haemoglobin  thus  obtained 
with  another  volume  of  water.     Treat  5  cc.  of  this  with  two  or 
three  drops  of  ammonium  sulphide  and  examine  immediately.     The 
colour  changes  to  a  red :  the  absorption  band  in  the  red  disappears, 
and  the  spectrum  of  oxyhaemoglobin  is  seen.     Warm  the  solution 
to  50°  C.  and  allow  it  to  stand  for  a  short  time  (possibly  with  the 
addition  of  another  drop  or  two  of  the  reducing  reagent).     The  two 
bands  give  place  to  the  single  band  of  reduced  haemoglobin.     Shake 
with  air :  oxyhaemoglobin  is  reformed. 

300.  Take  a  few  cc.  of  defibrinated  blood  in  a  test-tube,  add 
an  equal  quantity  of  water  and  warm  to  50°  C.  to  lake  the  blood. 
Oxygenate    the   solution  by   shaking  with    air,    adding    a   drop 
of  caprylic  alcohol  to  prevent  undue  frothing.     To  the  solution 
thus  obtained  add  an  equal  bulk  of  saturated  potassium  ferri- 
cyanide.    Mix  by  giving   one  shake,  and  then  hold  the  tube  at 
rest  in  an  oblique  position  for  a  short  time.     Note  the  bubbles  of 
gas  (oxygen)  that  are  evolved. 

NOTES. — i.  Oxyhaemoglobin  is  converted  into  methaemoglobin  by  the 
action  of  oxidising  reagents,  such  as  ferricyanides,  nitrites,  chlorates,  and 
permanganates,  and  in  the  body,  by  the  action  of  many  aromatic  substances, 
such  as  phenol. 


CH.  X.]  HAEMATIN.  247 

2.  The  reaction  is  peculiar  in  that  an  amount  of  oxygen  is  evolved 
equivalent  to  that  held  in  combination  by  the  oxyhaemoglobin,  although 
methaemoglobin  contains  the  same  percentage  of  oxygen  as  oxyhaemoglobin. 
This  reaction  is  the  basis  of  the  modern  method  of  estimating  the  amount  of 
oxygen  in  the  blood. 

The  reaction  might  be  represented  by  the  following  equation : — 

Oxyhaemoglobin.       From  ferricyanide  Methaemoglobin.  Gas 

and  water. 

The  oxygen  is  represented  as  being  in  a  different  state  of  combination  in 
methaemoglobin,  since  it  cannot  be  removed  by  submitting  the  compound  to  a 
vacuum. 

3.  When  methaemoglobin  is  treated  with  a  reducing  reagent,  the  first 
change  that  occurs  is  that  the  linkage  of  the  oxygen  atoms  reverts  to  that  of 
oxyhaemoglobin;  later,  the    oxygen  is  removed  and  reduced  haemoglobin 
formed. 

301.  Acid  haematin.      To  5  cc.  of  water  add  four  drops  of 
defibrinated  blood  and  five  drops  of  strong  acetic  acid  and  heat. 
The  colour  changes  to  brown  ;  and  the  solution  shows  an  absorption 
band  in  the  red,  which  is  further  from  the  D  line  than  that  of 
methaemoglobin.     Its  centre  is  about  X  650. 

302.  Acid  haematin  in  ethereal  solution.     Treat  a  few  cc. 
of  defibrinated  blood  with  one  drop  of  strong  hydrochloric  acid  and 
a  few  cc.  of  acetic  acid :  extract  this  with  about  5  cc.  of  ether  by 
gentle  shaking,  pour  the  ether  into  a  clean  tube  and  examine  it  with 
the  spectroscope.     There  is  a  prominent  band  in  the  red  (centre  X 
638) ;  on  dilution  with  ether  three  other  bands  can  be  seen  ;    a  very 
narrow  one  with  centre  X  582 ;    a  broad  one  stretching  from  about 
^  555  to  X  530  and  another  from  X  512  to  X  498. 

303.  Alkaline  haematin.    To  5  cc.  of  alcohol  add  3  drops  of  40 
per  cent,  soda  and  2  drops  of  defibrinated  blood.     Mix  and  heat  till 
the  alcohol  just  boils.     Add  an  equal  volume  of  water  and  examine 
spectroscopically.     A  prominent  band  is  seen  in  the  red,  stretching 
from  X  620  to  X  595.     On  the  blue- ward  side  is  a  shading  extending 
to  about  X  570.     The  blue  end  of  the  spectrum  may  be  absorbed. 

304.  Alkaline  haematin  in  alcohol.     Mix  defibrinated  blood 
into  a  thin  paste  with  solid  potassium  carbonate  and  evaporate  to 
complete  dryness  on  a  water  bath.     Powder  the  residue,  boil  with 


248  THE  RED  BLOOD  CORPUSCLES.  [CH.  X. 

alcohol  in  a  flask  on  the  water  bath  and  filter.  The  solution  con- 
tains alkaline  haematin  free  from  proteins.  It  shows  the  absorption 
band  of  alkaline  haematin  more  distinctly  than  the  solution 
prepared  in  Ex.  303. 

305.  Haemochromogen    (reduced   alkaline   haematin).     Pre- 
pare a  solution  of  alkaline  haematin  from  dilute  oxyhaemoglobin 
as  in  Ex.  303.     Treat  5  cc.  with  a  few  drops  of  ammonium  sulphide. 
The  colour  of  the  solution  changes  to  red.     Examine  with  the 
spectroscope.     Two  absorption  bands  are  seen  in  the  green.     The 
band  nearer  the  D  line  (the  a  band)  is  very  prominent  and  sharply 
denned,  with  its  centre  at  about  X  558.     The  /3  band  is  much  fainter 
and  has  its  centre  at  X  520. 

306.  To  5  cc.  of  water  add  one  drop  of  defibrinated  blood  and 
three  or  four  drops  of  ammonium  sulphide.     Mix  and  warm  cau- 
tiously till  the  oxyhaemoglobin  has  been  completely  reduced.     Add 
a  few  drops  of  40  per  cent,  soda  and  note  the  instantaneous  forma- 
tion of  haemochromogen. 

NOTE. — In  very  dilute  solutions  only  the  a  band  can  be  seen.  The 
absorption  of  light  in  this  region  is  so  intense  that  if  a  solution  of  oxyhaemo- 
globin, so  dilute  that  its  absorption  bands  cannot  be  readily  seen,  be  converted 
by  appropriate  means  into  haemochromogen,  the  a  band  of  this  pigment  is 
usually  observable. 

307.  Acid  haematoporphyrin.      To  a  few  cc.  of  concentrated 
sulphuric  acid  in  a  test-tube  add  two  drops  of  defibrinated  blood 
(see  note  to  Ex.  308)  and  mix  by  gentle  shaking.     Note  the  rich 
purple  colour  of  the  solution.      Examine  with  the  spectroscope. 
Two  bands  are  seen  :  the  a  band,  with  centre  at  X  600,  being  fainter 
and  narrower  than  the  /3  band,  centre  X  554. 

308.  Alkaline  haematoporphyrin.      To  the  solution  of  acid 
haematoporphyrin  just  prepared  add  five  or  six  more  drops  of 
defibrinated  blood,  shaking  gently  after  the  addition  of  each  drop. 
Pour  the  strong  solution  into  about  50  cc.  of  cold  water  in  a  beaker, 
stir  well  and  note  the  precipitate  that  rises  to  the  surface.     Transfer 
this  precipitate  to  a  test-tube  by  means  of  a  rod ;  treat  it  with  a  few 
cc.  of  alcohol  and  boil.    Add  5  cc.  of  sodium  hydroxide.   A  solution 
of  alkaline  haematoporphyrin  is  thus  obtained,  which  examined 


CH.  X.]  HAEMIN.  249 

spectroscopically.,  after  suitable  dilutions,  shows  a  four-banded 
spectrum.  The  centres  of  the  bands  are  at  X  622,  X  576,  X  539  and 
X  504  approximately. 

NOTE. — The  conversion  of  blood  pigment  into  haematoporphyrin  involves 
two  processes.  Firstly,  the  acid  splits  off  the  protein  constituent  (globin)  and 
forms  acid  haematin ;  secondly,  the  acid  haematin  loses  its  iron  and  becomes 
haematoporphyrin.  The  first  change  is  effected  very  readily  even  by  dilute 
acids,  but  the  separation  of  the  iron  from  the  haematin  normally  requires 
highly  concentrated  mineral  acids.  It  has,  however,  been  shown  by  Laidlaw 
that  if  the  blood  be  first  reduced  the  iron  is  split  off  with  much  greater  ease  by 
the  acid.  An  efficient  method  of  reducing  defibrinated  blood  is  that  of  "  auto- 
reduction,"  in  which  a  tightly  corked  vessel  full  of  blood  is  allowed  to  stand  for 
a  few  days.  If  Exercises  307  and  308  be  carried  out  with  this  reduced  blood, 
care  being  taken  by  use  of  a  pipette  to  prevent  re-oxidation,  the  haemoglobin  is 
entirely  converted  into  haematoporphyrin,  no  trace  of  the  brown  haematin 
being  left. 

It  sometimes  happens  that  on  pouring  the  acid  solution  into  water  a 
precipitate  is  not  obtained.  In  such  cases  it  is  necessary  to  repeat  the  experi- 
ment, but  to  pour  the  acid  haematoporphyrin  into  a  smaller  volume  of  water. 
On  making  a  portion  of  this  alkaline  with  40  per  cent,  soda  the  spectrum  of 
alkaline  haematoporphyrin  can  generally  be  observed.  It  is  sometimes 
necessary  to  examine  such  a  solution  in  a  thick  layer,  as  in  a  beaker. 

309.    Preparation  of  haemin  crystals. 

A  small  drop  of  blood  is  spread  to  form  a  film  on  a  glass  slide 
and  slowly  evaporated  till  it  is  quite  dry.  To  the  film  add  two  drops 
of  a  o-i  per  cent,  solution  of  potassium  chloride  in  glacial  acetic 
acid.  Cover  with  a  slip  and  heat  over  a  very  small  flame  till  bubbles 
appear  and  the  solution  is  boiling.  Immediately  allow  a  drop  or  two 
more  of  the  reagent  to  run  under  the  cover  slip  and  examine  under  a 
microscope. 

NOTE. — Haemin  is  the  di-acetyl  ester  of  haematin  hydrochloride.  The 
above  method  is  an  extremely  simple  one  of  obtaining  good  specimens  of  the 
crystals,  the  production  of  which  was  formerly  used  as  a  test  for  blood  in  cloth, 
etc.  It  is  important  not  to  burn  the  blood  during  the  drying  process,  and  also 
to  be  sure  that  the  acetic  solution  is  rapidly  brought  to  the  boiling  point. 

The  test  can  be  applied  to  dilute  solutions  of  haemoglobin  by  acidifying 
with  acetic  acid,  precipitating  with  freshly  prepared  tannic  acid,  and  treating 
the  dried  precipitate  in  a  slide  as  described  above.  Suspected  blood  stains  on 
linen,  instruments,  etc.,  should  be  extracted  with  a  little  alkali,  the  solution 
evaporated  to  dry  ness  and  treated  as  above. 

D.    Blood  constituents  and  their  analysis. 

Glucose.  Human  blood  contains  about  o-i  per  cent, 
of  glucose.  The  available  evidence  indicates  that  this 
exists  in  a  free  state  in  the  blood,  and  that  it  is  equally 


250  THE   BLOOD.  [CH.  X. 

distributed  between  the  plasma  and  the  corpuscles.  The 
concentration  in  the  blood  increases  after  the  ingestion  of 
considerable  amounts  of  glucose  or  cane  sugar,  but  only 
increases  after  a  meal  of  starch  if  the  subject  is  abnormal. 
The  extent  to  which  the  blood  sugar  rises  after  taking 
sugars  depends  on  the  rate  at  which  the  tissues,  especially 
the  liver,  can  assimilate  the  carbohydrate.  If  the  blood 
sugar  rises  beyond  0-12  per  cent,  the  condition  is  known  as 
hyperglycaemia.  This  is  generally  followed  by  the  excre- 
tion of  easily  detectable  amounts  of  glucose  in  the  urine,  or 
glycosuria,  the  severity  of  which  varies  with  the  degree  of 
hyperglycaemia,  and  also  on  the  permeability  of  the 
kidney  to  glucose.  It  must  be  noted  that  in  certain 
individuals  the  kidney  is  abnormally  permeable  to  sugar, 
so  that  marked  glycosuria  may  exist  without  hyper- 
glycaemia. This  condition  is  known  as  "renal  diabetes," 
or  "diabetes  innocens,"  this  latter  term  being  applied 
because  it  is  not  associated  with  the  evil  effects  of  the  other 
types  of  diabetes.  The  rate  at  which  the  liver  can  assimi- 
late sugar,  or  the  "tolerance  for  sugar/'  is  partially  depend- 
ent on  the  activities  of  various  organs,  such  as  the  pituitary, 
suprarenal  and  thyroid  glands.  Tolerance  is  best  deter- 
mined by  blood  analyses  at  appropriate  intervals  after 
administration  of  glucose  :  the  amount  in  the  urine  being 
dependent  on  the  renal  permeability. 

310.  Detection  of  glucose  in  blood.  Into  a  small  flask 
measure  15  cc.  of  distilled  water.  Add  3  cc.  of  fresh  defibrinated 
blood  obtained  from  a  slaughter  house.  Mix  to  lake  the  corpuscles. 
Heat  to  boiling,  and  keep  the  fluid  boiling  for  a  few  seconds.  Add 
4  cc.  of  1-25  per  cent.  "  colloidal  "  ("  dialysed  ")  iron,  adding  it  drop 
by  drop  and  constantly  agitating  the  flask.  The  proteins  are 
completely  precipitated.  Add  a  "  knife  point  "  of  solid  potassium 
sulphate  (to  precipitate  any  excess  of  iron),  and  shake  till  it  has 
dissolved.  Filter  through  a  small  paper.  To  5  cc.  of  the  clear  filtrate 
apply  Cole's  test  for  glucose  (Ex.  104).  A  distinct  positive  test  is 
obtained.  To  another  portion  of  the  filtrate  apply  the  picric  acid 
test  (Ex.  108),  controlling  the  latter  by  doing  a  blank  test  with  an 
equal  volume  of  water. 


CH.  X.]  BLOOD  SUGAR.  251 

NOTES. — i.  It  is  instructive  to  perform  similar  experiments  on  blood 
which  has  been  incubated  at  37°  C.  for  24  hours,  toluol  being  added  as  an 
antiseptic.  It  will  be  found  that  the  sugar  has  disappeared,  the  "  glycolysis  " 
being  due  to  the  action  of  special  oxidising  enzymes. 

2.     For  the  action  of  colloidal  iron  in  precipitating  proteins  see  page  10. 

311.    The  estimation  of  sugar  in  blood  (Benedict's  method).* 

Principle.  The  blood  is  laked,  treated  with  picric,  acid  and 
filtered.  An  aliquot  part  of  the  protein-free  filtrate  is  heat  eg!  with 
alkali.  The  glucose  reduces  some  of  the  picric  acid  to  picramic  acid 
(Ex.  1 08),  the  amount  of  which  is  estimated  in  a  colorimeter  against 
a  suitable  standard. 

Solutions  and  Apparatus  required: 

1.  Picric-picrate  mixture.     To  125  cc.  of  N.  soda  in  a  i  litre  measuring 
flask  add  about  700  cc.  of  hot  distilled  water,  and  then  36  grams,  of  pure,  dry 
picric  acid.     Wash  in  with  a  little  water,  shake  at  intervals  till  dissolved,  cool 
thoroughly,  make  up  to  i  litre  with  distilled  water,  mix  and  filter  if  the  solution 
is  not  crystal  clear. 

It  is  important  to  use  pure  dry  picric  acid.  It  is  advisable  to  recrystallise 
the  picric  acid  of  commerce  from  boiling  water,  filtering  the  hot  saturated 
solution  through  a  hot  water  funnel.  The  cold  solution  is  filtered  through 
a  Buchner  funnel  and  the  crystals  spread  out  on  layers  of  filter  paper  in  a 
warm  room  till  quite  dry.  The  mother  liquors  can  be  used  for  the  preparation 
of  the  stock  solution  of  glucose  mentioned  below.  It  must  be  remembered 
that  picric  acid  is  an  explosive,  and  that  it  is  not  safe  to  grind  it  in  a  mortar, 
or  to  send  it  through  the  post  without  damping  it. 

2.  Standard  solution  of  glucose.     The  stock  solution  required  is  a  i  per 
cent,  solution  in  saturated  picric  acid.     This  can  be  prepared  by  dissolving 
i   gram,  of  pure  glucose  (previously  dried  in  a  vacuum    desiccator)  in  cold 
saturated  picric  acid  and  making  the  volume  up  to  100  cc.  with  the  same 
solution.     Or  a  strong  (10  per  cent.)  solution  can  be  estimated  accurately 
by  means  of  a  polarimeter  and  a  volume  that  contains  exactly  i  gram,  diluted 
to  make  100  cc.  with  the  saturated  picric  acid. 

From  this  stock  (which  keeps  for  a  considerable  time)  is  prepared  daily  a 
solution  which  contains  0-64  mg.  in  i  cc.,  by  measuring  3-2  cc.  into  a  50  cc. 
flask  and  making  up  to  the  mark  with  distilled  water. 

An  alternative  standard  is  prepared  from  pure  picramic  acid,  should 
this  be  available.f  The  stock  solution  contains  100  mg.  of  picramic  acid  and 
200  mg.  of  anhydrous  sodium  carbonate  per  litre.  126  cc.  of  this  solution  are 
treated  with  i  cc.  of  20  per  cent,  sodium  carbonate  solution  and  15  cc.  of  the 
picric-picrate  mixture  and  diluted  to  300  cc.  with  distilled  water.  This 
solution  exactly  matches  the  colour  from  0-64  mg.  of  glucose  when  treated 
in  the  way  described  below  and  diluted  to  make  12-5  cc.  The  picramic  acid 
standard  is  not  heated  with  the  blood  sugar. 

*  Journ.  of  Biological  Chemistry,  xxxiv.,  p.  203  (1918). 

t  For  a  method  of  preparation  see  Egerer,  Journ.  of  Biological 
Chemistry,  xxxv.,  p.  565. 


252  THE  BLOOD.  [CH.  X. 

3.  Sodium  carbonate  solution.     Dissolve  200  grams,  of  pure  anhydrous 
sodium  carbonate  in  hot  distilled  water  and  make  the  volume  up  to  i  litre. 
Filter  when  cold.     The  solution  should  be  kept  in  a  warm  room,  as  it  may 
crystallise  out  if  the  temperature  gets  very  low. 

4.  Test-tubes    graduated    at    12-5  "and    25   cc.      A    suitable   internal 
diameter  is  f  inch.     It  is  convenient  to  have  one  tube  engraved  with  "  S  " 
for  the  standard  and  one  with  "  B  "  for  the  blood. 

5.  A  25  cc.  volumetric  flask.     If  this  is  not  to  hand,  one  of  the  graduated 
tubes  can  be  used. 

6.  Ostwald  pipettes  of  i  and  2  cc.     (See  fig.  48.) 

7.  A  colorimeter.     (See  p.  384.) 

Method.  2  cc.  of  blood  are  drawn  into  an  Ostwald  pipette, 
containing  a  trace  of  powdered  potassium  oxalate  to  prevent  coagu- 
lation. The  blood  must  be  drawn  under  strict  antiseptic  precau- 
tions. Discharge  the  blood  into  a  25  cc.  volumetric  flask  (or  gradu- 
ated tube),  and  wash  the  pipette  out  twice  with  distilled  water, 
adding  the  washings  to  the  flask.  After  standing  for  a  couple  of 
minutes,  with  gentle  agitation,  to  lake  the  blood,  add  the  picric- 
picrate  mixture  to  the  mark,  and  thoroughly  shake  the  mixture. 
After  standing  for  a  minute  or  two*  the  mixture  is  poured  on  to  a 
dry  filter  and  the  clear  filtrate  collected  in  a  small  dry  flask.  Measure 
8  cc.  of  the  filtrate  into  one  of  the  "  B  "  calibrated  tubes,  add  i  cc. 
of  the  sodium  carbonate  solution,  mix,  and  plug  with  cotton  wool. 
If  glucose  is  being  used  as  a  standard,  measure  i  cc.  of  the  diluted 
solution  mentioned  above  (i.e.  0-64  mg.  glucose)  into  one  of  the 
"  S  "  tubes,  using  an  Ostwald  pipette.  Add  3  cc.  of  distilled  water, 
4  cc.  of  the  picric-picrate  mixture  and  i  cc.  of  the  sodium  carbonate. 
Mix  and  plug  with  cotton  wool.  Have  a  beaker  or  can  of  water 
boiling.  Immerse  both  tubes  in  this  and  note  the  time.  After 
exactly  ten  minutes'  heating  in  the  bath,  the  water  of  which  must  be 
kept  boiling  the  whole  time,  remove  the  tubes  and  cool  thoroughly. 
Dilute  the  standard  to  the  12-5  cc.  mark  with  distilled  water. 
Dilute  the  "  B  "  tube  to  the  same  mark  and  compare  the  colours  of 
the  two  solutions.  If  that  of  "  B  "  is  much  greater  than  that  of 
"  S,"  the  "  B  "  tube  can  be  treated  with  2-5,  5,  7-5,  10,  or  12-5  cc. 
of  water,  until  the  colours  of  the  two  solutions  appear  to  be  about 
the  same,  making  a  note  of  the  amount  of  water  added.  The  same 

*  The  method  can  be  interrupted  at  this  stage,  the  picric  acid  preventing 
glycolysis. 


CH.  X.]  BLOOD   SUGAR.  253 

procedure  is  adopted  if  the  standard  solution  of  picramic  acid  is 
used.  The  two  solutions  are  now  compared  in  a  colorimeter  (see 
p.  385),  the  standard  being  set  at  15  mm. 

Calculation.     2  cc.  of  blood  are  taken,  diluted  to  25  cc.  and  8  cc. 
of  the  nitrate  taken.     The  amount  of  blood  actually  used  for  the 

o 

test  is  therefore  —  x  2  =  0-64  cc. 
25 

Since  the  colorimeter  readings  are  inversely  proportional  to 
the  concentrations  of  glucose 

mg.  glucose  in  0-64  cc.  blood  Reading  of  "  S  " 


0-64  (i.e.  mg.  glucose  in  standard)         Reading  of  "  B  " 

"S"  0-6, 
r7BT'  X  o^ 
Reading  of  "  S  " 


c      .  ,,,      ,      Reading  of  "S"      0-64 

So  glucose  in  I  cc.  of  blood  =  -        — x  -    -  mg. 

Reading  of  "  B  "      0-64 


So  gram,  of  glucose  in  100  cc.  blood  = 


Reading  of  "  B  "  x  10 

This  is  for  a  dilution  of  "  B  "  to  12-5  cc.     Should  the  dilution 
exceed  this,  a  correction  must  be  applied.     Thus,  if  7-5  cc.  of  water 
are  added  beyond  the  12-5  mark,  the  result  must  be  multiplied  by 
12-5  +  7'5  =   ™_  =  I>6 
12-5          12 -5 

NOTE. — Benedict  thinks  that  in  advanced  nephritis  it  might  be  necessary 
to  modify  the  procedure  owing  to  the  presence  of  interfering  substances,  such 
as  creatinine.  His  original  paper  should  be  consulted  for  details. 

312.    The  micro-analysis  of  sugar  in  blood  by  Bang's  method. 

Principle.  A  few  drops  of  blood  are  drawn  up  on  a  piece  of 
absorbing  paper,  the  amount  taken  being  determined  by  measure- 
ment or  by  weighing  with  a  torsion  balance.  After  drying,  a  boiling 
acidified  solution  of  potassium  chloride  is  added.  The  proteins  are 
coagulated  and  the  glucose  that  diffuses  out  of  the  paper  is  estimated 
by  an  indirect  method. 

Solutions  and  Apparatus  required. 

i.  Stock  copper  solution.  In  a  litre  flask  place  700  cc.  of  boiled  out, 
cold  distilled  water.  Warm  to  about  30°  C.  Add  160  grams,  of  pure  powdered 
potassium  bicarbonate.  When  dissolved,  add  66  grams,  of  pure  potassium 
chloride.  Cool  and  add  100  grams,  of  potassium  carbonate.  Then  100  cc.  of 
a  4 -4  per  cent,  solution  of  pure  crystalline  copper  sulphate.  Allow  to  stand 


254 


THE    BLOOD. 


[CH.  X. 


for  a  while  and  fill  up  to  the  mark  with  boiled  out  distilled  water.  Vigorous 
shaking  of  the  fluid  must  be  avoided.  The  solution  should  not  be  used  for  24 
hours. 

2.  Acidified  potassium  chloride.     To  1360  cc.  of  a  cold  saturated  solution 
of  pure,  recrystallised  potassium  chloride  add  3-75  cc.  of  N.  hydrochloric  acid, 
and  make  the  volume  up  to  2  litres  with  distilled  water. 

3.  Soluble  starch,     i  gram,  of  soluble  starch  is  dissolved  in  100  cc.  of  a 
boiling  solution  of  potassium  chloride,  saturated  at  room  temperature.     After 
cooling  the  volume  is  made  up  to  100  cc.  with  distilled  water. 

4.     iodine.      To  2  grams,  of  potassium  iodide  in  a  100  cc.  volumetric 

200 

flask  add  i  to  2  cc.  of  a  2  per  cent,  solution  of  potassium  iodate  and  a  few  cc. 
of  distilled  water.  Then  add  5  cc.  of  o-i  N.  hydrochloric  acid  by  means  of  a 
pipette  and  make  the  volume  up  to  100  cc.  with  cold,  recently  boiled,  distilled 
water. 

5.  Absorbing  papers.      A  thick  blotting  paper 
cut  into  pieces   about    25  x  8    mm.      The    paper 
used  must  be  quite  pure,  and  not  yield  any  soluble 
matter  on  extraction. 

6.  An  accurately  graduated  pipette  to  contain 
o-i  to  0-13  gram,  of  distilled  water  at  15°  C.     These 
pipettes  are  best  made  from  millimetre  tubing  and 
should  be  engraved  with  the  exact  weight  of  water 
they  deliver.     The  weight  of  blood  used  is  obtained 
by  multiplying  the  weight  of  water  by  i  -06.     If  a 
torsion  balance  (p.  388)  is  available  the  measure- 
ment of  the  blood  is  simplified. 

7.  A   standard  heating  apparatus  (see  fig.  15, 
p.  136).     The  pressure  of  gas  and  height  of  gauze 
must  be  so  arranged  that  14  cc.  of  the  acidified 
potassium  chloride  solution  are  brought  to  the  boil- 
ing point  in  i£  minutes  ±  5  sees. 

8.  50   cc.  flask    of    "  Duro  "   glass,    with    a 
straight  neck,  so  that  a  piece  of  thick  walled  rubber 
tubing   about   3    mm.  thick,  and  about  5  cm    in 
length,  can  be  slipped  over  the  neck.     A  screw  clip 

is  fitted,  so  that  the  flask  can  be  hermetically  sealed.  This  is  shewn  in 
fig.  3i. 

9.  Apparatus  for  titration  in  an  atmosphere  of  CO2.     Owing  to  the 
rapidity  with  which  the  cuprous  chloride  is  oxidised  it  is  necessary  to  exclude 
air  by  means  of  an  atmosphere  of  CO2.     This  is  best  accomplished  by  means 
of  the  simple  apparatus  shewn  in  fig.  32. 

10.  Cylinder  of  CO2,  or  a  Kipp's  generating  apparatus  charged  with 
pieces  of  marble  and  hydrochloric  acid  (i  in  3). 

n.     A  steam  oven  for  drying  the  paper.     A  large  cork  is  transfixed  with 
a  needle  or  pin,  on  which  the  clip  that  holds  the  paper  can  be  suspended. 

Method. 

i.     To  obtain  a  measured  amount  of  blood. 


Fig.  31.      Flask   fitted 
for  sugar  estimation. 


A.     By  weighing  with  a  torsion  balance. 


CH.  X.] 


BANG'S  METHOD. 


255 


A  paper  is  held  in  a  small  spring  clip  and  the  clip  and  paper 
are  weighed  together  as  described  on  p.  388. 

The  hand  of  the  subject  is  washed  in  warm  water  and  dried. 
The  subject  is  instructed  to  swing  the  arm  backwards  and  forwards, 
keeping  the  hand  as  low  as  possible.  The  finger  is  pricked  with  a 
sterile  bayonet-pointed  probe  on  the  back  of  the  finger  about 
J  inch  above  the  nail.  A  piece  of  rubber  tubing  is  wound  tightly 
round  the  middle  joint  of  the  finger.  On  firmly  flexing  the  finger 


Fig.  32.     Apparatus  for  titration  in  an  atmosphere  of  CO2. 

A.  Wash  bottle  containing  water. 

B.  Tube  fitted  into  flask  so  that  the  iodine  can  fall  from  burette  directly 
into  the  fluid. 

the  blood  usually  wells  up  in  sufficient  amount.  The  blood  is  taken 
up  on  the  paper,  until  the  paper  is  fairly  covered.  It  is  undesirable 
to  have  the  paper  fully  saturated  with  blood.  The  paper  and  clip 
are  immediately  weighed  on  the  torsion  balance,  and  the  weight  of 
blood  taken  is  thus  known.  A  convenient  amount  is  about  120  mg. 

B.     By  means  of  a  pipette  (see  6  above). 

The  pipette  being  cleaned  and  dried,  and  the  absorbing  paper 
ready,  the  finger  is  pricked  as  described  above.     When  a  large  drop 


256  THE    BLOOD.  [CH.  X. 

of  blood  has  collected  the  pipette  is  placed  in  this  and  very  gentle 
suction  applied,  the  pipette  being  held  nearly  horizontal,  and  care 
being  taken  to  avoid  the  introduction  of  an  air  bubble.  If  the  blood 
does  not  reach  the  mark,  the  finger  is  firmly  pressed  again,  and  the 
blood  drawn  slightly  above  the  mark.  The  exterior  of  the  pipette 
is  wiped  with  a  piece  of  filter  paper,  the  blood  run  on  this  to  the  mark 
and  then  discharged  on  to  the  piece  of  absorbing  paper,  the  pipette 
being  completely  blown  out  at  the  end  of  the  operation. 

With  a  little  practice  the  whole  process  is  rapidly  completed,  and 
there  is  little  risk  of  the  blood  clotting.  As  soon  as  the  pipette  has 
been  drained  a  little  dilute  alkali  should  be  sucked  up  and  blown 
out  two  or  three  times,  to  prevent  the  film  of  blood  clotting  in  the 
pipette.  This  is  then  thoroughly  washed  with  distilled  water, 
alcohol  and  then  with  ether.  It  can  be  dried  by  attaching  it  to  a 
suction  pump. 

2.  Drying  the  paper.     After  standing  for  a  few  minutes  the 
paper  is  held  in  a  little  clip  and  suspended  in  the  steam  oven  at 
100°  C.  for  3  to  4  minutes. 

3.  Coagulation  of  the  proteins.      Place  the  paper,  without  the 
clip,  in  a  clean,  dry,  rather  wide  test-tube  of  such  a  size  that  the 
paper  is  completely  immersed  by  the  6J  cc.  of  solution.     Measure 
6J  cc.  of  the  acid  solution  of  potassium  chloride  into  another  test- 
tube.     Boil  and  rapidly  pour  the  vigorously  boiling  solution  on  to 
the  paper  in  the  other  tube.     Allow  the  tube  to  stand  for  at  least 
30  minutes.     Transfer  the  fluid  to  the  50  cc.  flask.     Wash  the  paper 
with  another  6J  cc.  of  cold  acid  potassium  chloride,  and  add  this  to 
the  fluid  in  the  flask. 

4.  Method  of  heating  the  solution.     To  the  flask  add  i  cc.  of 
the  copper  solution  and  fit  the  rubber  sleeve  on  to  the  neck.     Place 
the  screw  clip  over  the  rubber,  but  do  not  tighten  it.     Have  the 
heating  apparatus  ready  and  properly  adjusted.     Place  the  flask 
on  the  centre  of  the  heated  gauze  and  note  the  time.     The  solution 
should  commence  to  boil  in  i|  minutes.     Allow  the  solution  to  boil 
for  exactly  2  minutes.     Just  before  the  two  minutes  is  completed, 
commence  to  tighten  the  screw  clip  C.     When  the  time  has  expired 
tighten  the  clip  very  firmly,  remove  the  flask  at  once  and  plunge  it 


CH.  x.]  BANG'S  METHOD.  257 

into  cold  water.     Allow  it  to  cool  in  a  stream  of  water  for  at  least 
i  minute. 

NOTE. — A  special  pair  of  forceps  has  been  devised  for  holding  the  flask 
and  sealing  the  rubber  tube. 

5.  Titration  of  the  fluid.  Loosen  the  clip,  remove  the  rubber, 
and  immediately  fit  in  the  tube  from  the  CO2  apparatus,  the  gas 
having  previously  been  turned  on.  Add  3  or  4  drops  of  the  starch 
solution  and  titrate  with  N/2OO  iodine  from  a  microburette.  The 
titration  is  completed  when  the  "  starch-blue  "  tint  persists  for 
about  20  seconds. 


6.     Calculation. 

cc.  of  iodine  —0-16  cc. 


=  mg.  of  glucose  in  the  blood  taken. 


7.     Example. 

Weight  of  blood  =  118  mg. 
Iodine  required  =  0-68  cc. 

0-68  —  0-16      0-52  ,  ,  T      , 

-  =  -   -  =  0-13  mg.  glucose  in  118  mg.  blood. 

4  4 

=  o-ii  per  cent. 

NOTES. — i.  The  results  obtained  are  apt  to  be  slightly  higher  than  those 
by  Benedict's  method,  but  the  advantage  of  a  "  finger-prick  "  method  of 
drawing  the  blood  is  so  great  that  it  overweighs  the  possible  slight  error.  It 
is  most  important  to  use  the  purest  chemicals  obtainable  and  to  pay  strict 
attention  to  details. 

2.  Bang  has  recently  published  a  new  method  of  analysis  (see  p.  126,  9), 
but  the  author,  having  failed  to  get  consistent  results  with  it,  prefers  to  use  a 
method  with  which  he  is  familiar. 

313.  The  micro-analysis  of  chlorides  in  blood  by  Bang's 
method. 

Principle.  A  few  drops  of  blood  are  taken  up  from  a  finger 
prick  on  to  a  piece  of  absorbing  paper.  The  proteins  are  coagulated 
by  pouring  on  a  boiling  acid  solution  of  magnesium  sulphate.  After 
cooling,  2  cc.  of  standard  silver  nitrate  are  added  and  the  silver 
chloride  filtered  off,  a  little  kieselgur  being  added  to  aid  filtration. 
The  silver  nitrate  in  the  filtrate  is  treated  with  2  cc.  of  a  standard 
solution  of  potassium  iodide  and  potassium  iodate  and  a  few  drops 

s 


258  THE   BLOOD.  [CH.  X. 

of  starch  solution.  The  iodate  yields  free  iodine  owing  to  the 
presence  of  the  acid  in  the  coagulating  fluid.  The  mixture  is  then 
titrated  with  standard  silver  till  the  blue  colour  disappears.  From 
the  amount  of  silver  required  to  effect  this,  the  amount  of  silver  in 
the  filtrate  can  be  determined.  Thus  the  amount  of  silver  that  has 
disappeared  in  the  formation  of  silver  chloride  can  be  calculated, 
and  so  the  amount  of  chloride  in  the  blood  taken. 

Preparation  of  reagents. 

1.  N/ioo  silver  nitrate.     1-7  gram,  of  pure  silver  nitrate  are  dissolved  in 
distilled  water  and  the  volume  made  up  to  i  litre.     It  should  be  stored  in  a 
dark  bottle  and  kept  in  the  dark. 

2.  Iodide  andiodate  solution.  0-015  gram,  of  potassium  iodate  and  i  -7  gram, 
of  potassium  iodide  are  dissolved  in  water  and  the  volume  made  up  to  i  litre. 
2  cc.  of  the  solution  are  measured  and  treated  with  10  cc.  of  solution  3  and  a 
few  drops  of  the  soluble  starch.     This  mixture  is  titrated  with  N/ioo  silver 
nitrate  from  a  microburette  until  the  blue  colour  disappears.     The  strength 
of  the  solution  must  be  adjusted  by  the  addition  of  water  or  of  a  dilute  solution 
of  potassium  iodide  until  2  cc.  require  exactly  2  cc.  of  the  silver  nitrate. 

3.  Acid  magnesium  sulphate.     2  litres  of  a  30  per  cent,  solution  of 
magnesium  sulphate,  120  cc.  of  strong  pure  nitric  acid  (sp.  gr.i'42)  and  280  cc. 
of  distilled  water  are  mixed. 

4.  Starch  solution,    i  gram,  of  soluble  starch  are  suspended  in  a  little  cold 
water  and  poured  into  about  80  cc.  of  boiling  distilled  water.   20  grams,  of  pure 
potassium  nitrate  are  added  to  the  mixture.     The  solution  is  poured  into  a 
number  of  small  sterile  bottles  and  stoppered  whilst  still  hot.     In  this  way  the 
solution  can  be  preserved  for  a  considerable  time. 

5.  Kieselgur.     This  should  be  purified  by  heating  to  a  dull  red,  washing 
with  10  per  cent,  acetic  acid  and  then  with  distilled  water  and  heating  again 
to  redness. 

Measure  0-2  cc.  of  distilled  water  into  a  tube  about  5  cm.  in  length  and 
about  5  mm.  bore  which  has  one  end  sealed.  Make  a  mark  with  a  file  or  a 
label  to  show  the  height  of  the  fluid.  Dry  the  tube  thoroughly.  The  amount 
of  kieselgur  for  each  experiment  is  measured  by  filling  the  tube  to  the  mark 
after  gently  tapping. 

Since  kieselgur  adsorbs  silver  nitrate  it  is  essential  to  determine  the 
amount  of  this  for  every  specimen  by  a  blank  experiment  conducted  as 
follows  :  To  10  cc.  of  solution  3  add  2  cc.  of  the  silver  nitrate  and  the  measured 
amount  of  kieselgur.  Shake  and  filter  through  a  Gooch  crucible  as  described 
below.  Wash  the  tube  and  the  kieselgur  twice  with  5  cc.  of  distilled  water. 
To  the  filtrate  add  2  cc.  of  the  solution  2  and  a  few  drops  of  the  starch  solution. 
Titrate  cautiously  with  N/ioo  silver  nitrate  from  a  microburette  till  the  blue 
colour  is  discharged.  The  amount  of  silver  required  corresponds  to  the 
amount  adsorbed  by  the  kieselgur. 

Method  of  Analysis. 

i.  To  obtain  a  measured  amount  of  blood.  The  procedure 
is  exactly  the  same  as  described  in  the  section  on  the  micro-analysis 
of  sugar  (p.  254). 


CH.  X.] 


CHLORIDES. 


259 


2.  Coagulation  of  the  proteins.  Place  the  paper  in  a  clean,  dry, 
rather  wide  test-tube.  Into  another  clean  tube  measure  10  cc.  of 
the  acid  magnesium  sulphate  and  boil.  Whilst  vigorously  boiling 
pour  the  solution  on  to  the  paper  and  allow  it  to  stand  for  30  minutes. 

3.  Removal  of  the  silver  chloride.  To  the  tube,  still  containing 
the  paper,  add  2  cc.  of  N/ioo  silver  nitrate  and  the  measured  amount 
of  kieselgur.  Grease  the  rim  of  the  tube  with  a 
smear  of  vaseline  and  shake  vigorously.  Filter 
through  pa  6  cc.  Gooch  crucible  into  a  125  cc. 
filtering"~flask  connected  to  a  water  pump  (see 
fig-  33)-||The  bottom  of  the  crucible  is  covered 
with  a  piece  of  filter  paper  cut  a  trifle  larger  than 
the  bottom.  The  paper  is  then  washed  with  a 
little  water  which  is  sucked  through.  Care  must 
be  taken  to  see  that  all  the  perforations  of  the 
crucible  are  covered.  Empty  the  flask  and  before 
turning  on  the  pressure  fill  the  crucible  with  the 
mixture  in  the  tube,  being  careful  to  get  as  much 
kieselgur  as  possible  into  the  crucible.  Allow  a 
few  drops  to  filter  through  before  turning  on  the 
pressure.  Filtration  is  rapid  and  the  nitrate 
is  usually  quite  clear.  If  it  is  cloudy  it  must  be 
refiltered. 


Fig.  33.  Gooch 
crucible  and  fil- 
tering apparatus 
for  micro-analysis 
of  chlorides. 


4.  Washing  the  paper.     When  the  whole  of  the  fluid  has  been 
filtered  add  5  cc.  of  distilled  water  to  the  tube,  shake  vigorously, 
pour  it  into  the  crucible  and  filter.     Repeat  this  operation  once 
more. 

5.  Titration  of  the  silver.    To  the  fluid  in  the  flask  add  2  cc. 
of  the  iodide-iodate  solution  and  a  few  drops  of  the  starch  solution. 
Titrate  against  a  white  ground  with  N/ioo   silver  nitrate  from 
a  icroburette.     The   blue   colour  gradually  fades   as   the   silver 
iodide  is  formed  and  then  sharply  disappears,  leaving  a  yellowish 
green  solution. 

6.  Calculation  of  results.     2  cc.  of  N/ioo  silver  =  2  cc.  of  the 
iodide-iodate  solution.    The  volume  of  N/ioo  silver  required  to  effect 
the  disappearance  of  the  blue  colour  is  the  volume  of  silver  that  has 


260  THE   BLOOD.  [CH.  X. 

disappeared  from  the  tube.  Of  this  volume  a  certain  amount  has 
been  adsorbed  by  the  kieselgur.  The  remainder  has  formed  silver 
chloride  with  the  chlorides  of  the  blood.  Since  I  cc.  of  N/ioo  silver 
=  0-585  mg.  NaCl,  the  amount  of  NaCl  in  the  blood  taken  can  be 
calculated. 

7.     Example. 

Weight  of  blood  taken  =  116  mg. 

Volume  of  N/ioo  silver  adsorbed  by  kieselgur  =0-12  cc. 

Volume  of  N/ioo  silver  required  for  titration  =  1*21  cc. 

Volume  of  N/ioo  silver  precipitated  as  silver  chloride 
=  i-2i  -  0-12  =  i -09  cc. 

NaCl  in  116  mg.  blood  =  1-09  x  0-585  =  0-638  mg. 
NaCl  per  cent.  =  0-55. 

314.    The  estimation  of  the  non-protein  nitrogen  of  blood. 

Principle.  Blood  is  treated  with  water  and  then  with  a  solution 
of  metaphosphoric  acid,  which  precipitates  the  whole  of  the  proteins 
(Ex.  17).  An  aliquot  portion  of  the  nitrate  is  concentrated  and  the 
total  nitrogen  determined  by  Kjeldahl's  method. 

Reagents  and  Apparatus  Beguiled. 

1.  The  usual  requisites  for  Kjeldahl's  method  (see  p.  322). 

2.  Freshly    prepared    solution    of   metaphosphoric    acid.     Weigh    out 
2 -5  grams,  of  "  glacial  phosphoric  acid,"  and  crush  it  in  a  mortar  with  8  cc.  of 
distilled  water. 

3.  The  apparatus  shewn  in  fig.  34.     A  is  a  wash  bottle  containing  I  in  5 
sulphuric  acid,  to  remove  ammonia  from  the  air. 

B.  A  large  boiling  tube  (9  x  j"\  in.). 

C.  A  funnel,  with  tap  and  side  piece,  as  shewn. 
E.    A  plain  condenser. 

G.    A  boiling-tube  (10  x  ij  in.),  with  the  flange  ground  off  on  a  wet  stone. 
The  tubes  B  and  G  and  the  inner  tube  of  E  must  be  of  resistance  glass. 

4.  A  micro-burner,   or,   better,   an   ordinary   Bunsen   fitted    with    a 
rose- top. 

5.  A  foot  bellows  or  a  blast  pump  to  enable  the  final  titration  to  be 
conducted  in  a  CO2-free  atmosphere.     The  air  is  led  through  wash  bottles 
containing  i  in  4  sulphuric  acid  (to  remove  ammonia)  and  then  through  soda 
(to  remove  the  CO2).     It  then  passes  to  a  thick- walled  tube,  which  is  bent  to 
fit  the  tube  G,  as  shewn  in  fig.  35. 


CH.  X.] 


NON-PROTEIN    NITROGEN. 


26l 


Method.  To  about  20  cc.  of  distilled  water  in  a  50  cc.  volumetric 
flask  add  5  cc.  of  recently  drawn  blood  and  mix  by  gentle  agitation. 
Add  3  cc.  of  the  freshly  prepared  solution  of  metaphosphoric  acid 
and  mix.  Full  up  to  the  mark  with  distilled  water,  mix  and  transfer 
the  contents  to  a  100  cc.  flask.  Shake  vigorously  at  intervals  for  at 
least  5  minutes,  or  allow  the  mixture  to  stand  for  a  longer  period. 


Fig-  34- 


Apparatus  for  Micro- Kjeldahl  by 
Cole's  modification. 


Tube  for 
titrating  in  a  CO. 
free  atmosphere. 


Filter  into  a  dry  tube  through  a  dry  paper.  The  filtrate  should  be 
crystal  clear ;  if  it  is  not,  it  must  be  passed  through  the  filter  till 
clear.  Transfer  10  cc.  of  the  filtrate  to  a  boiling  tube  (about 
7x1  in.),  add  2  cc.  of  pure  sulphuric  acid  and  cautiously  concen- 
trate to  a  small  volume  over  a  free  flame.*  This  is  a  rather  tedious 


*  See  Langstroth,  Journ.  Biol.  Chem.,  xxxvi.,  p.  377. 


262  THE    BLOOD.  [CH.  X. 

operation.  A  test-tube  holder  should  be  improvised  by  holding  a 
piece  of  folded  paper  round  the  neck  of  the  tube.  This  is  held 
obliquely  and  shaken  vigorously  during  the  boiling.  In  this  way 
loss  by  spurting  can  be  avoided.  When  the  fluid  has  been 
concentrated  to  about  4  cc.,  another  10  cc.  of  the  nitrate  is 
added  and  the  greater  part  of  the  fluid  boiled  off  again.  When 
the  volume  has  been  reduced  to  about  3  cc.  add  i  gram,  of  pure 
potassium  sulphate  and  2  drops  of  saturated  copper  sulphate. 
Heat  over  a  micro-burner,  using  a  Folin's  fume-absorber  (p.  383). 
A  very  small  flame,  not  quite  touching  the  tube,  is  better  than  a 
large  flame  a  few  inches  away.  A  screen  to  keep  off  draughts  is 
sometimes  necessary.  After  a  time  the  fluid  goes  nearly  black,  and 
subsequently  lightens  in  colour  until  it  is  a  faint  blue  or  green. 
Heating  must  be  continued  for  at  least  5  minutes  after  this  stage 
has  been  reached. 

Remove  the  flame  and  allow  to  cool  until  the  tube  can  be  held 
in  the  hand.  Then  add  10  cc.  of  distilled  water  and  shake.  If  a  solid 
cake  separates  out,  the  tube  must  be  cautiously  heated  until  this  has 
completely  dissolved.  Transfer  the  contents  to  tube  B  of  the 
apparatus  shewn  in  fig.  34.  Wash  out  the  tube  with  another  10  cc. 
of  distilled  water,  adding  this  to  B.  Wash  out  with  10  cc.  of  rectified 
alcohol  and  connect  up  to  the  apparatus.  Into  tube  G  measure 
10  cc.  of  standard  sulphuric  acid  (about  0-04  N.),  add  3  or  4  drops  of 
methyl  red  and  connect  the  tube  up  to  the  apparatus,  the  tube  being 
immersed  in  a  tall  jar  of  cold  water.  Connect  G  to  a  suction  pump 
as  shewn,  and  connect  the  condenser  E  to  the  water  supply  by  the 
lower  end.  Into  C  place  some  40  per  cent,  soda,  which  has  been  well 
boiled  in  open  dishes  to  remove  any  ammonia.  Start  the  pump  so 
that  a  gentle  stream  of  air  passes  through  the  apparatus.  Allow 
some  of  the  soda  to  run  into  the  solution  until  it  becomes  alkaline, 
as  will  be  seen  by  the  fluid  becoming  blue.  Heat  the  solution  with  a 
micro-burner,  or  a  Bunsen  with  a  rose-top,  to  boiling  point.  The 
use  of  a  rose-top  minimises  the  risk  of  the  tube  cracking.  The  flame 
should  not  play  directly  on  the  glass.  The  air  current  and  the 
boiling  can  be  regulated  to  minimise  the  risk  of  splashing.  The 
distillation  should  be  allowed  to  continue  for  10  to  n  minutes. 
Remove  the  flame,  but  do  not  stop  the  air  current.  Disconnect 
the  rubber  joint  at  D  and  wash  the  inner  tube  of  the  condenser 


CH.  X.]  NON-PROTEIN    NITROGEN.  263 

down  into  G  with  a  jet  of  water.  Disconnect  the  pump  and  then 
the  rubber  connexion  of  F  to  the  condenser.  Remove  the  stopper 
of  G  with  the  tubes  and  wash  down  the  interior  and  exterior  of  F 
into  G.  Fit  the  tube  shewn  in  fig.  35  to  G  and  titrate  with 
standard  CO2-free  soda  (which  should  be  about  0-04  N.). 

Calculation  and  Example  (see  p.  323). 

Soda  was  0-032  N. 

Acid  was  0-029  N.   (i   cc.    =  0-029  x  14  =  0-406  mg.  N.). 

Soda  required  for  titration  was  7-61  cc.  =  76-1  x  —  =8-40  cc.  of  the  acid2 

So  acid  neutralised  by  the  Nitrogen  in  20  cc.  filtrate  was  10  -  8-4  =  1-6  cc. 

So  non-protein  N.  in  2  cc.  blood  =  1-6  x  0-406  =  0-65  mg. 

Blank  determination  =  0-02  mg.  N. 

So  non-protein  N.  in  2  cc.  blood  =  0-63  mg. 

Non -protein  N.  in  100  cc.  =  31-5  mg. 

NOTES. — i.  Folin  and  Denis  (Journ.  Biol.  Chtm.,  xxvi.,  p.  491)  introduced, 
the  method  of  precipitating  the  proteins  with  metaphosphoric  acid.  They 
estimate  the  nitrogen  by  direct  Nesslerisation  after  incinerating  with  a  mixture 
of  sulphuric  and  phosphoric  acids.  The  author  has  not  been  successful  in 
repeating  their  results,  being  troubled  with  the  rapid  formation  of  a  cloud  after 
adding  Nessler's  reagent.  Under  these  circumstances  the  above  method  of 
distillation  of  the  ammonia  has  been  elaborated. 

2.  The  amount  of  non-protein  nitrogen  in  normal  human  blood  seems 
to  vary  between  30  and  50  mgms.  per  100  cc.  It  is  increased  in  certain  types 
of  nephritis  and  in  severe  hepatic  disease. 


CHAPTER   XI. 


THE   CONSTITUENTS  OF   BILE. 


Bile  is  secreted  continuously  into  the  hepatic  ducts 
by  the  liver.  During  the  intervals  of  digestion  it  is  stored 
in  the  gall  bladder,  being  poured  into  the  duodenum  when 
the  acid  chyme  passes  through  the  pylorus. 

During  its  stay  in  the  gall  bladder  there  is  an  absorp- 
tion of  water  and  an  increase  in  the  protein  content, 
resulting  in  an  increase  in  the  specific  gravity  from  about 
1010  to  1040. 

The  percentage  composition  of  human  bile  varies 
considerably.  The  following  are  average  figures  : — 


Water 
Solids 

Bile  salts 

Protein 

Bile  pigments 

Cholesterol 

Lecithin  and  fat 

Inorganic  salts 


The  bile  salts  are  the  sodium  salts  of  glycocholic 
and  taurocholic  acids.  They  are  formed  by  the  condensa- 
tion of  cholalic  acid  (C24H4O5)  with  glycine  (amino-acetic 
acid,  CH2.NH2.COOH)  and  taurine  respectively.  Glycine 
is  one  of  the  products  obtained  by  the  hydrolysis  of 
proteins. 


From 

From 

Gall  Bladder. 

Fistula. 

86 

..  98 

14 

.  .     2 

9 

..   0-8 

3 

..   0-3 

O-2 

.  .   0-06 

I  -O      .  . 

.  .   0-04 

0-8    .. 

..   0-8 

CH.  XI.]  BILE   SALTS.  265 

Taurine  is  derived  from  a  similar  product,  cysteine. 
CH2SH  CH2.SO3H 

CH.NH2  ->     CH2.NH2 

COOH 

Cysteine.  Taurine. 

The  bile  acids  are  hydrolysed  into  their  constituents 
by  boiling  acids  and  also  by  the  intestinal  bacteria. 

The  bile  salts  are  soluble  in  water  and  alcohol,  in- 
soluble in  ether. 

Their  solutions  have  a  remarkably  low  surface  tension. 
(See  Hay's  test.) 

They  have  the  following  functions  :— 

1 .  They  have  a  marked  adjuvant  action  on  pancreatic 
lipase.     (See  Ex.  167.) 

2.  They  are  solvents  for  the  fatty  acids  and  markedly 
increase  the  absorption  of  fats. 

3.  They   thus   help   to   remove   the   fatty  film   sur- 
rounding the  protein,  and  allow  the  proteolytic  ferments 
to  act.     In  this  way,  by  assisting  the  absorption  of  pro- 
teins, they  diminish  bacterial  decomposition.     They  are 
not  direct  antiseptics. 

Preparation  of  Bile  Salts. — Mix  40  cc.  of  ox  gall  with  enough  animal 
charcoal  (about  10  grams.)  to  form  a  paste.  Evaporate  to  dryness  over  a 
water  bath,  stirring  at  intervals.  Grind  the  residue  in  a  mortar,  transfer 
it  to  a  flask,  add  about  70  cc.  of  96  per  cent,  or  absolute  alcohol  and  boil 
on  the  water  bath  for  20  minutes.  Cool  and  filter  into  a  dry  beaker.  Add 
ether  to  the  filtrate  till  there  is  a  slight  permanent  cloudiness.  Cover  the 
beaker  with  a  glass  plate  and  allow  it  to  stand  in  a  cool  place  for  24  hours. 
A  crystalline  mass  of  bile  salts  separates  out.  The  crystals  are  filtered  off  and 
allowed  to  dry  in  the  air. 

For  the  following  tests  use  diluted  ox  or  sheep  gall : — 

315.    Pettenkofer's  test  for  bile  salts.      To   5  cc.   of  the 

solution  add  a  small  particle  of  cane-sugar  and  shake  or*  warm  till 
this  has  completely  dissolved.  To  the  cooled  solution^add  5  cc. 
of  concentrated  sulphuric  acid,  inclining  the  test-tube  so  that  the 


266  THE   CONSTITUENTS   OF   BILE.  [CH.  XI. 

acid  settles  to  the  bottom.     Gently  shake  the  test-tube  from  side  to 
side.     As  the  fluids  gradually  mix  a  deep  purple  colour  develops. 

NOTES. — i.  This  reaction  depends  on  the  production  of  furfurol  from  the 
cane-sugar  by  the  strong  acid.  (See  Ex.  114.) 

2.  If  too  much  cane-sugar  be  taken  the  fluid  will  turn  brown  or  black, 
owing  to  the  charring  produced. 

3.  Proteins  give  a  very  similar  reaction  with  furfurol  in  the  presence  of 
strong  acids.     (See  Ex.  26.)     Proteins  also  tend  to  give  a  brown  char  with 
sulphuric  acid.     For  these  reasons  it  is  advisable  to  remove  the  proteins  from 
solution  before  attempting  the  test. 

4.  The  purple  colour  obtained  is  only  stable  in  the  presence  of  strong 
sulphuric  acid.     It  disappears  on  dilution  with  water. 

5.  If  a  small  portion  of  the  coloured  fluid  be  diluted  with  50  per  cent, 
sulphuric  acid,  and  examined  with  the  spectroscope,  two  absorption  bands  will 
be  seen,  one  between  the  lines  C  and  D,  nearer  the  latter ;    the  other  in  the 
green,  overlapping  E  and  B. 

6.  The  test  cannot  be  applied  directly  to  urine,  owing  to  the  presence  of 
chromogenic  substances  that  yield  intense  colours  with  sulphuric  acid. 

316.  Hay's  test  for  bile  salts.      Take  10  cc.  of  the  solu- 
tion in  a  test-tube.     Sprinkle  the  surface  with  flowers  of  sulphur  and 
note  that  they  fall  through  the  liquid  to  the  bottom  of  the  tube. 
Repeat  the  test  with  water,  noting  that  the  particles  remain  on  the 
surface. 

NOTES. — i.  This  test  for  bile  salts  depends  on  the  remarkable  property 
that  they  possess  of  lowering  the  surface  tension  of  water,  thus  enabling  the 
particles  of  sulphur  to  sink  through  the  fluid. 

2.  The  test  is  of  great  value  for  the  detection  of  bile  salts  in  urine. 

3.  This  property  of  bile  salts  is  utilised  by  draughtsmen  in  preparing 
tracings  on  oiled  paper,  on  which  ink  collects  in  drops,  and  does  not  spread 
well.     If  the  paper  be  first  treated  with  a  little  ox  gall  and  allowed  to  dry  the 
difficulty  is  removed,  owing  to  the  reduction  in  surface  tension. 

4.  A  method  for  estimating  bile  salts  in  urine  has  been  described  by 
Grunbaum,  depending  on  this  property.     The  rate  of  escape  of  the  urine  from 
standard  capillary  tubes  is  noted,  the  rate  increasing  with  the  concentration  of 
bile  salts. 

317.  Oliver's  test  for  bile  salts.    Acidify  5  cc.  of  the  solution 
with  two  or  three  drops  of  strong  acetic  acid,  filtering  if  necessary. 
To  the  acid  solution  add  an  equal  quantity  of  i  per  cent,  solution  of 
peptone.     A  white  milkiness  or  a  decided  precipitate  is  produced, 
insoluble  in  excess  of  acid. 

NOTES. — i.  The  precipitate  formed  consists  of  a  compound  of  protein 
with  bile  acids. 

2.     The  test  can  be  applied  to  urine.     (Ex.  379.) 


CH.  XI.]  BILE   PIGMENTS.  267 

The  Bile  Pigments. 

Bilirubin,  C32H36N4O6,  is  a  reddish-brown  pigment 
most  abundant  in  the  bile  of  carnivora.  It  is  readily 
oxidised  by  the  oxygen  of  the  air  into  biliverdin,  C32H36N4O8, 
the  green  pigment  found  mostly  in  the  bile  of  herbivora. 
These  compounds  are  formed  in  the  liver  cells  from  the 
products  of  disintegration  of  haemoglobin.  Haematin  is 
C»HttN/)4Fe,  and  haematoporphyrin  is  isomeric  with 
bilirubin. 

They  are  weak  acids,  forming  sodium  and  calcium 
salts,  the  latter  being  insoluble  in  water.  Free  bilirubin 
is  soluble  in  ether  and  chloroform  :  the  sodium  compound 
is  insoluble,  as  is  free  or  combined  biliverdin. 

By  oxidation  bilirubin  is  converted,  through  a  num- 
ber of  ill-defined  bodies,  such  as  bilicyanin,  and  bilifuscin, 
into  choletelin,  the  end  product  of  Gmelin's  reaction. 

By  further  oxidation  a  compound,  haematinic  acid 
(C8H8O5),  is  formed,  identical  with  the  product  obtained  by 
the  oxidation  of  haematin  or  haematoporphyrin. 

By  reduction  with  sodium  amalgam  in  alcoholic  solu- 
tion the  bile  pigments  are  converted  into  hydrobilirubin, 
which  is  also  formed  by  the  action  of  more  powerful  reduc- 
ing agents  on  haematin  or  haematoporphyrin. 

These  facts  all  indicate  the  close  relationship  between 
haematin  and  the  bile  pigments. 

In  the  bowel  the  bacteria  first  reduce  bilirubin  to 
hydrobilirubin.  This  is  then  attacked,  two  nitrogen 
atoms  being  probably  removed,  the  result  being  the  for- 
mation of  urobilin,  which  is  mainly  excreted  in  the  faeces, 
being  sometimes  called  "  stercobilin."  A  certain  amount 
however,  is  absorbed  into  the  blood,  and  excreted  by  the 
liver  into  the  bile,  whilst  a  small  amount  is  excreted  by 
the  kidney  in  the  form  of  urobilinogen.  (See  p.  278.) 

318.    Huppert-Cole  test  for  bile  pigments. 

Boil  about  15  cc.  of  the  fluid  in  a  test-tube.  Add  two  drops  of  a 
saturated  solution  of  magnesium  sulphate,  then  add  a  10  per  cent. 


268  THE    CONSTITUENTS   OF   BILE.  [CH.  XI. 

solution  of  barium  chloride,  drop  by  drop,  boiling  between  each 
addition.  Continue  to  add  the  barium  chloride  until  no  further 
precipitate  is  obtained.  Allow  the  tube  to  stand  for  a  minute. 
Pour  off  the  supernatant  fluid  as  cleanly  as  possible  or  use  a  centrifuge . 
To  the  precipitate  add  3  to  5  cc.  of  97  per  cent,  alcohol,  two  drops  of 
strong  sulphuric  acid,  and  two  drops  of  a  5  per  cent,  aqueous  solu- 
tion of  potassium  chlorate.  Oil  for  half  a  minute  and  allow  the 
barium  sulphate  to  settle.  The  presence  of  bile  pigments  is  indi- 
cated by  the  alcoholic  solution  being  coloured  a  greenish  blue. 

NOTES. — i.  To  render  the  test  more  delicate,  pour  off  the  alcoholic 
solution  from  the  barium  sulphate  into  a  dry  tube.  Add  about  one-third  its 
volume  of  chloroform  and  mix.  To  the  solution  add  about  an  equal  volume 
of  water,  place  the  thumb  on  the  tube,  invert  once  or  twice  and  allow  the 
chloroform  to  separate.  It  contains  the  bluish  pigment  in  solution. 

2.  The  bile  pigment  is  adsorbed  on  to  the  barium  sulphate  precipitate, 
but  passes  into  solution  again  in  acid  alcohol.     The  chlorate  acts  as  a  very 
weak  oxidising  reagent,  converting  bilirubin  and  biliverdin  to  the  characteristic 
blue  compound. 

3.  The  author  claims  that  it  is  a  very  much  more  delicate  test  than  the 
one  that  follows. 

319.  Gmelin's  test  for  bile  pigments.  Take  a  few  cc.  of 
fuming  yellow  nitric  acid  in  a  test-tube,  and  by  means  of  a  pipette 
carefully  place  on  the  surface  of  this  an  equal  amount  of  bile.  Shake 
the  tube  very  gently  from  side  to  side,  and  note  the  play  of  colours 
in  the  bile  as  it  becomes  oxidised  by  the  acid.  Proceeding  from 
acid  to  bile  the  colours  are  yellow,  red,  violet,  blue,  and  green. 

NOTES. — This  test  can  be  modified  in  many  ways. 

1.  Add  a  drop  of  yellow  nitric  acid  to  a  thin  film  of  bile  on  a  white 
porcelain  plate.     The  drop  of  acid  becomes  surrounded  by  rings  of  the  various 
colours. 

2.  Filter  some  diluted  bile  repeatedly  through  an  ordinary  filter  paper, 
and  then  place  a  drop  of  fuming  nitric  acid  on  the  paper.     The  play  of  colours 
is  usually  well  seen. 

Cholesterol  has  been  described  on  p.  161,  and  Lecithin 
on  p.  163. 

The  Protein  of  Bile. 

When  bile  is  treated  with  acetic  acid  a  precipitate  is 
formed  insoluble  in  excess.  This  was  formerly  thought 
to  be  mucin.  But  it  has  been  shown  that  it  is  nucleo- 
protein,  the  bile  salts  present  preventing  the  re-solution 


CH.  XI.]  THE   PROTEINS  OF   BILE.  269 

in  strong  acetic  acid.     (See  Ex.  3 1 7.)     In  human  bile,  how- 
ever, mucin  is  present  as  well  as  nucleoprotein. 

The  protein  is  secreted  by  the  cells  lining  the  ducts 
and  the  gall  bladder,  so  that  bile  from  the  gall  bladder 
contains  a  much  greater  percentage  than  fistula  bile. 

320.  To  a  small  quantity  of  undiluted  bile  add  strong  acetic 
acid,  drop  by  drop.  A  precipitate  is  formed,  insoluble  in  excess  of 
acid.  This  precipitate  consists  of  a  nucleoprotein,  together  with  a 
considerable  amount  of  the  bile  salts  and  bile  pigments. 


CHAPTER   XII. 


URINE    AND    ITS    CHIEF    CONSTITUENTS. 

A.    The  average  composition. 

The  composition  of  the  urine  varies  with  the  individual 
and  with  the  diet.  Below  we  given  the  figures  in  grams,  for 
the  daily  output  of 

A.  The  average  man  on  the  average  mixed  diet. 

B.  An  individual  on  a  liberal  diet. 

C.  The  same  individual  on  a  diet  deficient  in  proteins. 
B.  and  C.  are  taken  from  a  paper  by  Folin. 


A. 

B. 

C. 

Nitrogen. 

s-.  en 

0     ^H 

-M    ° 
Ij 

fc3 

*l 

Nitrogen. 

o^ 

^  ° 

h 

l->    05 
0)  4-> 

fk£ 
•^  r~" 

Nitrogen. 

H-.cn 

°* 
%K 

t-,  13 

X| 

Urea 

3° 

H 

87-5 

31-6 

14-7 

87-5 

4-72 

2'2 

61-7 

Ammonia 

0-6 

o-5 

3'i 

•6 

0-49 

3-0 

•51 

0-42 

n-3 

Creatinine 

i-55 

o-57 

3'6 

i-55 

0-58 

3'6 

1-61 

0-60 

17-2 

Uric  Acid 

0-7 

0-23 

1-4 

•54 

0-18 

i-i 

•27 

0-09 

2-5 

Undetermined 

0-7 

4'4 

0-85 

4-8 

0-27 

7'3 

Total—  N 

16-0 

1OO-O 

16-8 

100-0 

3-6 

IOO-O 

Inorganic  SO3 

2-92 

88-2 

3-27 

90-0 

0-46 

60.5 

Ethereal  SO3 

•22 

6-6 

0-19 

5'2 

o-io 

13-2 

Neutral  SO3 

•17 

5'2 

0-18 

4'8 

0'20 

26-3 

Total  SO3 

3'i 

IOO-O 

3-64 

100-0 

0-76 

lOO'O 

CH.  XII.]  SPECIFIC   GRAVITY.  271 

B.    The  Physical  Chemistry  of  the  Urine. 

/.     General  Properties. 

Normal  human  urine  is  a  clear  yellowish  fluid,  the 
depth  of  the  tint  depending  largely  on  the  concentration. 
On  standing,  a  cloud  (nubecula)  of  mucoid  containing 
epithelial  cells  separates  out.  After  a  heavy  meal  urine 
may  be  passed  cloudy,  due  to  earthy  phosphates  and 
carbonates.  On  standing,  these  settle  to  the  bottom  of 
the  vessel  as  a  white  deposit,  insoluble  on  warming,  but 
soluble  in  acids. 

Also  on  standing  a  cloud  of  urates  may  settle  as  a 
reddish  deposit  that  clears  up  on  warming. 

Fresh  urine  has  a  characteristic  odour  of  the  aromatic 
type,  due  to  the  presence  of  some  substance  that  has 
not  yet  been  recognised.  On  standing,  an  unpleasant 
ammoniacal  odour  develops  as  the  result  of  bacterial 
decomposition. 

//.     The  Specific  Gravity. 

Usually  lies  between  1012  and  1024  (water  =  1000). 
With  copious  drinking  it  may  fall  to  1002.  After  excessive 
perspiration  it  may  rise  to  1040. 

The  determination  of  the  specific  gravity  for  clinical 
purposes  is  most  conveniently  made  by  means  of  a  urino- 
meter,  a  weighted  cylinder  that  floats  in  the  urine.  The 
depth  to  which  it  sinks  depends  on  the  density  of  the 
fluid,  and  this  can  be  read  directly  by  means  of  a  graduated 
scale  on  the  stem.  The  instrument  is  calibrated  for  a 
certain  temperature,  usually  15°  C. 

The  urine  should  be  either  cooled  or  warmed  to  this 
temperature,  or  a  correction  made  by  adding  i  unit  for 
every  3  degrees  above  this,  or  subtracting  i  for  every  3 
degrees  below  the  standard.  Thus,  if  the  reading  be  1018 
at  1 8°  C.,  the  corrected  Sp.  Gr.  is  1019. 


272 


URINE. 


[CH.  XII. 


To  obtain  the  best  results  two  separate  instruments 
should  be  at  hand,  the  one  calibrated  from  1000  to  1020 
and  the  other  from  1020  to  1040. 

The  total  amount  of  solids  in  the  urine  can  be  roughly 
calculated  from  the  specific  gravity  by  Long's  coefficient.  The 
last  two  figures  of  the  specific  gravity  x  2-6  gives  total  solids 
in  1000  cc. 

Thus  specific  gravity  at  25°  C  =  1017. 

Total  solids  in  1000  cc.  =  17  x  2-6  =  44-2  grams. 

Haser's  coefficient  (2-33)  on  a  similar  basis,  but  calculated 
for  15°  C.  is  probably  inaccurate. 

321.  Take  the  specific  gravity  of  normal  urine  by 
means  of  a  urinometer.  Wipe  the  instrument  clean, 
and  float  it  in  the  centre  of  a  cylinder  containing  the 
urine.  Remove  all  froth,  by  means  of  filter  paper  or 
by  placing  a  single  drop  of  ether  on  the  surface  of  the 
urine.  Take  care  that  the  instrument  does  not  touch 
the  sides  of  the  vessel.  Place  the  eye  level  with  the 
surface  of  the  fluid  and  read  the  division  of  the  scale 
to  which  the  latter  reaches.  Read  the  level  of  the  true 
surface  of  the  urine,  not  the  top  of  the  meniscus 
around  the  stem. 

///.     The  Osmotic  Pressure  (Cryoscopy). 

The  method  of  taking  the  freezing  point  of 
a  fluid  is  described  on  p.  8,  and  the  subject  has 
been  considered  from  a  theoretical  standpoint 
on  p.  5. 

Flg-  36.  in  urine  the  concentrations  of  certain  sub- 

Urmometer.  stances^  such   as   urea^   are  much  greater  than 

they  are  in  the  blood.  The  work  done  by  the  kidney 
in  effecting  this  concentration  can  be  calculated  from  a 
consideration  of  the  osmotic  concentration,  i.e.  A,  of 
each  substance  in  blood  and  urine.  It  is  quite  erroneous 
to  imagine  that  the  work  done  can  be  calculated  from 
a  knowledge  of  the  total  osmotic  concentration  of  the 
blood  and  urine  respectively.  But,  at  the  same  time, 
the  determination  of  A  of  the  blood  and  of  the  urine 
secreted  by  each  kidney  in  certain  renal  diseases  may 


CH.  XII.]  REACTION.  273 

give  us  valuable  information  as  to  the  relative  activities 
of  the  two  organs. 

A  of  blood  is  about  0-55°  C.,  the  same  as  that  of  a  0*9 
per  cent,  solution  of  sodium  chloride. 

A  of  urine  varies  considerably  with  the  diet,  volume  of 
fluid  taken  and  other  conditions.  For  the  mixed  24  hours 
urine  of  an  average  man  it  is  usually  about  1-2°  C.  The 
following  values  are  of  interest  in  this  connection  : — 

A  x  volume  of  urine  =  molecular  diuresis. 

is  of  considerable  pathological  signi- 

NaCl  per  cent. 

ficance.  It  is  fairly  constant  in  health,  varying  between 
1*25  and  1-6.  It  exceeds  1-7  in  heart  disease  or  in  any 
condition  that  causes  a  retardation  of  the  renal  circulation. 
The  only  febrile  condition  in  which  it  is  less  than  1-7  is 
malaria. 


IV.     Reaction. 

Normal  human  urine  is  generally  acid  to  litmus,  the 
average  PH  of  24  hour  specimens  being  about  6-0.  The 
reaction  varies  with  the  diet,  being  greatest  on  a  meat  diet, 
owing  to  the  oxidation  (in  the  body)  of  the  sulphur  and 
phosphorus  to  sulphuric  and  phosphoric  acids.  On  a 
vegetable  diet,  however,  the  urine  may  become  alkaline, 
as  it  is  in  herbivora,  owing  to  the  organic  salts  being 
oxidised  to  alkaline  carbonates.  During  the  secretion  of 
the  acid  gastric  juice  into  the  stomach,  the  urine  may 
become  alkaline,  the  so-called  "  alkaline  tide." 

The  acid  reaction  of  the  urine  is  mainly  due  to  the 
excretion  of  acid  phosphates  and  of  weak  organic  acids.  A 
certain  amount  of  the  acids  produced  in  the  body  are 
neutralised  by  ammonia  and  excreted  as  ammonium  salts 
in  the  urine.  The  ingestion  of  acids  or  of  acid  phosphates 
usually  leads  to  an  increase  in  the  excretion  of  titratable 
acids  (or  acid  salts)  and  of  non-titratable  neutral  am- 


274 


URINE. 


[CH.  XII 


monium  salts.  The  sum  of  the  two  can  be  taken  as  a 
measure  of  the  total  amount  of  acid  eliminated  from  the 
body.  The  titratable  acid  excreted  is  usually  measured  by 
titrating  to  phenol  phthalein,  but  it  is  better  to  titrate  to 
blood  reaction,  i.e.  PH  =  7*45.  By  the  use  of  the  compara- 
tor described  below,  this  is  a  very  simple  matter. 

Palmer  and  Henderson*  have  studied  the  relationships 
between  reaction,  volume  of  urine,  etc.  The  following 
table  gives  some  of  the  results  obtained  in  apparently 
healthy  subjects  : — 


PH 

24  hours 
Volume  in  cc. 

Titratable 
Acid  in  cc.  of 
o-i  N  Acid. 

NH3  in  cc.  of 
o'i  N  Acid. 

A 

N 

A 

N 

A+N 

R 

5'4 

1026 

320 

367 

687 

0-87 

5'7 

H93 

303 

384 

687 

0-79 

6-0 

1259 

263 

365 

628 

0-72 

6-6 

I4OO 

224 

357 

58l 

0-63 

Av 

erage  of  a 

11  the  case 

S. 

5'94 

1231 

278 

370 

649 

0-75 

They  note  that 

1.  A  increases  with  the  hydrogen-ion  concentration. 

2.  With  constancy  in  the  excretion  of  phosphoric  acid 
the  hydrogen-ion  concentration  varies  with  A. 

3.  With  constancy  of  A,  the  hydrogen-ion  concentra- 
tion varies  as  the  phosphoric  acid. 

4.  N  is  fairly  constant.    Normally  the  final  regulation 
of  the  reaction  through  excretion  falls  upon  the  phosphates. 

5.  The  volume  increases  as  the  acidity  decreases. 
The  variations  in  these  various  factors  in  renal  disease 


*  Journ.  of  Biol.  Chem.,  xvii.,  p.  305. 


CH.  XII.] 


ACID    EXCRETION. 


275 


have  been  investigated  by  Palmer  and  Henderson,*  who 
find  that  in  certain  types  of  the  disease  ("High  Ratio") 
there  is  a  remarkable  increase  in  R,  mainly  due  to  a  deficit 
in  the  excretion  of  ammonia.  In  other  types  of  renal 
disease  the  various  factors  are  nearer  to  normal,  as  can 
be  seen  from  the  table  below. 


PH 

Vol- 
ume 

A 

N 

A+N 

R 

Type 

5-2 

1650 

3i8 

165 

483 

2-08 

High  Ratio 

5-2 

1086 

287 

276 

563 

1-03 

Medium  Ratio 

5-6 

1187 

244 

34i 

585 

0*72 

Low  Ratio 

It  would  seem  that  an  important  factor  in  renal  disease 
is  a  difficulty  in  the  excretion  of  ammonium  salts,  so  that 
to  maintain  the  normal  reaction  of  the  blood  the  kidney  is 
forced  to  excrete  an  abnormally  acid  urine.  This,  in  its 
turn,  may  cause  a  further  degeneration  of  the  renal  tissues. 

A  further  point  of  interest  in  connexion  with  the  acidity 
of  the  urine  is  that,  according  to  van  Slyke,  the  alkali 
reserve  of  the  body  can  be  determined  and  the  condition  of 
"acidosis"  diagnosed  from  the  indication  given  by  the 
analyses  described  below,  f 

The  method  of  determining  the  PH  of  urine  is  given  on 
p.  29. 

322.    The  estimation  of  titratable  acid  in  urine  (Cole's  method). 

Principle.  The  urine  is  titrated  to  PH  =  7-45  (the  average 
reaction  of  normal  blood),  using  a  large  form  of  Cole  and  Onslow's 


*  Journ.  of  Biol.  Chem.,  xxi.,  p.  37. 

f  A  full  account  will  be  found  in  the  following  papers : — Fitz  and  van 
Slyke,  Journ.  of  Biol.  Chem.,  xxx.,  p.  389.    Van  Slyke,  ibid,  xxxiii.,  p.  271. 


276 


URINE. 


[CH.  XII. 


comparator  (see  p.  21).  The  concentrations  of  the  indicator  in  the 
urine  and  the  two  buffer  solutions,  and  also  the  intensity  of  the 
pigment  in  the  three  tubes  containing  urine  are  kept  constant  by 
the  addition  of  equivalent  quantities  of  water  and  soda  respectively. 


Solutions  and  Apparatus  required. 

i .     A  comparator  for  holding  the  tubes  (see  fig.  37) .     This  should  be  fitted 
with  a  ground  glass  screen,  as  shewn  in  the  diagram. 


2.  Tubes  of  clear  resistance  glass  (7  x  i  in.)  to  fit  the 
comparator.  These  should  have  the  same  internal  diameter. 
They  can  be  calibrated  by  measuring  25  cc.  of  water  from  a 
pipette  into  a  number  and  selecting  those  tubes  in  which  the 
fluid  reaches  the  same  level.  A  better  method  is  to  use  a  hard 


Fig.  37.      Cole  and  Onslow's  Comparator  for  large  tubes. 

wood  gauge,  which  is  slightly  conical  and  is  marked  with  rings 
corresponding  to  every  1-64  in.  diameter.  The  gauge  is  pressed 
into  the  tubes  and  those  selected  of  the  same  internal  diameter. 
The  method  of  using  this  is  shewn  in  fig.  38.  It  is  convenient  to 
choose  sets  of  tubes  and  to  mark  each  member^of  a  set  with  a 
distinguishing  letter  by  means  of  a  diamond. 


Fig.  38- 
Gauge. 


3.  Buffer  solution,  PH  =  7'42-     Prepared  by  treating  50  cc.   of  0-2  M. 
KH2PO4  with  the  equivalent  of  39-9  cc.  of  0-2  N.NaOH  and  diluting  to  make 
200  cc.  with  distilled  water  (see  p.  27). 

4.  Buffer    solution,     PH  =  7-47.      Prepared    as    above,    but    use    the 
equivalent  of  40*2  cc.  of  0*2  N.NaOH. 

5.  Standard  alkali,  see  p.  380.     It  is  convenient  to  use  o-i  N.  from  an 
ordinary  burette  or  0*2  N.  from  a  microburette. 


CH.  XII.]  ESTIMATION   OF    TITRATABLE   ACID.  277 

Method. 

Measure  20  cc.  of  the  urine  into  tubes  (2),  (3)  and  (6). 

Measure  20  cc.  of  the  buffer  PH  =  7*42  into  (i). 

Measure  20  cc.  of  the  buffer  PH  =  7-47  into  (5). 

Place  about  20  cc.  of  water  into  (4). 

Add  10  to  15  drops  of  0-02  per  cent,  phenol  red  to  (i),  (3)  and 
(5),  using  a  dropping  pipette  (fig.  5)  and  adding  exactly  the  same 
amount  to  each  tube. 

Mix  the  contents  of  the  tubes  by  smartly  rotating  them  be- 
tween the  palms  of  the  hands. 

Titrate  (3)  with  the  standard  soda.  A  precipitate  of  earthy 
phosphates  may  appear  and  the  colour  as  seen  through  Y  gradually 
approaches  that  seen  through  X.  Let  the  amount  of  soda  added 
be  (a)  cc. 

To  (i)  and  (5)  add  (a)  cc.  of  distilled  water  from  a  burette  or 
pipette. 

To  (2)  and  (6)  add  (a)  cc.  of  the  standard  soda.  Read  the 
burette. 

Complete  the  titration  of  (3)  until  the  colour  as  seen  through 
Y  is  intermediate  between  that  seen  through  X  and  Z.  The  tubes 
must  be  spun  just  before  the  observation  is  made  to  ensure  an  equal 
distribution  of  any  precipitate.  Let  the  amount  of  soda  required 
for  this  operation  be  (b)  cc. 

Calculation. 

20  cc.  of  urine  require  (a)  +  (b)  cc.,  say  (A)  cc.  of  the  soda, 
which  is  (c)  times  Normal. 

So  100  cc.  require  (A)  x  5  cc.  of  the  soda. 

So  100  cc.  contain  (A)  x  5  x  10  x  (c)  cc.  of  o-i  N.  acid. 

NOTES. — i.  It  is  important  that  the  examination  be  made  in  fresh 
urine.  Should  this  be  impossible  a  little  toluol  should  be  added  to  the  speci- 
men to  prevent  the  ammoniacal  fermentation  of  the  urea. 

2.  Should  the  urine  be  so  concentrated  that  a  precipitate  of  urates 
separates  out,  the  urine  may  be  diluted  with  an  equal  volume  of  distilled 
water  and  the  mixture  gently  warmed  till  it  clears.     It  is  then  cooled  under 
the  tap  and  the  estimation  made  as  described  above,  allowance  being  made  for 
the  dilution  in  the  final  calculation. 

3.  By  a  similar  method,  enzyme  solutions,  digestion  mixtures,  etc.,  can 
be   brought   to   any   desired   hydrogen-ion   concentration.     Suitable   buffer 
solutions  and  indicators  can  be  prepared  according  to  the  directions  given 
in  pages  22  to  28. 


278  URINE.  [CH.  XII. 

C.     The  Pigments  of  Urine. 

Urochrome  is  the  chief  pigment  of  normal  urine. 
It  is  a  yellow  substance  which  has  no  definite  absorption 
band.  Nothing  certain  is  known  as  to  its  constitution  or 
origin,  except  that  it  is  apparently  not  derived  from  the 
bile  pigments.  It  has  marked  reducing  properties. 

Urobilin  occurs  in  fresh  normal  urine  as  its  chromo- 
gen,  urobilinogen.  This  is  converted  into  urobilin  by  acids 
or  by  the  action  of  light  and  oxygen.  The  amount 
excreted  is  markedly  increased  in  fevers,  in  diseases  of 
the  liver  and  bile  passages,  by  destruction  of  the  red 
corpuscles,  especially  in  pernicious  anaemia  and  malaria, 
and  during  the  absorption  of  blood  clots.  In  certain  of 
these  cases  the  urobilin  itself  is  found  in  the  urine,  and  can 
be  identified  by  its  characteristic  absorption  band,  urobilin- 
ogen not  giving  a  definite  band. 

Urobilinogen  is  a  pyrrol  body  and  is  responsible  for 
Ehrlich's  reaction  with  ^-dimethyl-amino-benzaldehyde. 

The  origin  of  urobilin  from  the  bile  pigments  is  dis- 
cussed on  page  267.  It  may  be  added  that  the  urobilin 
absorbed  from  the  bowel  into  the  circulation  is  mostly 
excreted  by  the  liver  into  the  bile,  so  that  only  a  small 
portion  reaches  the  urine.  Should  the  liver  cells  be 
injured,  or  should  there  be  any  interference  with  the 
circulation  through  the  liver,  there  is  a  considerable  increase 
in  the  excretion  of  either  urobilin  or  urobilinogen  in  the 
urine. 

If  the  common  bile  duct  is  completely  occluded  by  a 
gall  stone  or  by  a  growth  the  urobilin  and  urobilinogen  are 
absent  from  the  urine  and  faeces.  Should  the  obstruction 
be  removed  there  is  often  a  period  during  which  the 
amounts  of  these  substances  in  the  urine  is  exceptionally 
large. 

Uroerythrin  is  found  in  small  amounts  in  normal 
urine.  It  is  increased  in  fever  and  certain  diseases  of  the 
liver. 


CH     XII.]  UROBILIN.  279 

It  is  soluble  in  amyl  alcohol.  Solutions  have  a  reddish 
colour,  but  are  unstable  to  light. 

The  pigment  is  usually  associated  with  the  urates  or 
uric  acid  of  the  urine. 

Haematoporphyrin  is  found  in  traces  in  normal  urine. 
There  is  a  certain  increase  in  fevers,  and  some  other 
diseases,  but  a  very  marked  increase  in  certain  cases  of 
poisoning  by  sulphonal  or  trional,  especially  in  women. 

Urorosein  occurs  in  urine  as  a  chromogen  which  is 
converted  into  the  pigment  by  the  action  of  strong  acids, 
such  as  hydrochloric  and  sulphuric. 

It  is  insoluble  in  ether  and  is  thus  distinguished  from 
indigo  blue  formed  in  the  test  for  indican.  (Ex.  318.) 

The  chromogen  seems  to  be  an  indol  body,  possibly 
indol-acetic  acid. 

323.  Note  the  colour  of  normal  urine  and  examine  some  in  a 
beaker  by  the  spectroscope.     Note  that  there  are  no  definite  ab- 
sorption bands,  but  a  general  absorption  of  the  violet.     Urochrome, 
the  chief  urinary  pigment,  yields  no  bands. 

324.  Saturate   at   least   200   cc.   of  urine  with   ammonium 
sulphate.     Filter  off  the  precipitate  and  let  it  dry  completely  in  the 
air.     Extract  it  with  a  small  amount  of  strong  alcohol.     A  brownish 
solution  containing  urobilinogen  is  obtained.     Treat  this  with  a  few 
drops   of   hydrochloric   acid :     the  urobilinogen   is   converted   to 
urobilin.      Examine   with   the   spectroscope,    and   note   a   single 
absorption  band  situated  at  the  junction  of  the  blue  and  the  green. 
Its  centre  is  about  A  490. 

325.  Bogomolow's  test  for  urobilin  or  urobilinogen.     Treat 
10  cc.  of  the  urine  with  10  drops  of  20  per  cent,  copper  sulphate. 
Add  about  4  cc.  of  chloroform,  place  the  thumb  on  top  of  the  tube 
and  invert  10  times  without  shaking.     If  abnormal  amounts  of 
urobilin  or  urobilinogen  are  present,  the  chloroform  layer  is  coloured 
yellow. 

Place  the  finger  on  the  upper  end  of  a  dry  5  cc.  pipette  and 
insert  the  lower  end  into  the  chloroform  layer.  Suck  up  the  chloro- 


280  URINE.  [CH.  XII. 

form  solution  and  transfer  it  to  a  dry  tube.  The  chloroform  usually 
separates  as  a  clear  fluid,  which  is  of  a  faint  pink  colour  if  urobilin 
is  present.  A  characteristic  absorption  band,  with  centre  about 
X  500  can  be  seen. 

326.  Schlesinger's  test  for  urobilin.  To  10  cc.  of  urine 
add  3  drops  of  a  5  per  cent,  alcoholic  solution  of  iodine  (  to  convert 
urobilinogen  to  urobilin).  Into  another  test:tube  place  I  gram,  of  zinc 
acetate  and  10  cc.  of  absolute  alcohol.  Mix  the  two  solutions  and 
repeatedly  decant  until  all  the  zinc  acetate  has  dissolved.  Filter. 
Examine  the  filtrate  in  a  test-tube,  16  mm.  wide,  in  daylight  falling 
from  behind  the  observer.  A  green  fluorescence  is  seen  if  urobilin 
or  urobilinogen  are  present. 

NOTE. — The  above  method  is  a  modification  introduced  by  Marcussen 
and  Hansen  (Journ.  Biol.  Chem.,  xxxvi.,  p.  381).  They  state  that  ammoniacal 
urines  should  be  acidified  with  acetic  acid.  They  find  that  in  patients  suffering 
from  liver  complaints  they  can  detect  the  fluorescence  when  the  urine  has  been 
diluted  40  to  80  times,  and  are  of  the  opinion  that  unless  it  can  be  detected  in  a 
dilution  of  r  in  20  a  pathological  urobilinuria  has  not  been  definitely  established. 

D.    The  Inorganic  Constituents. 

Rations. 

Sodium  and  potassium  are  found  to  the  extent  of 
3-2  gram.  K2O  and  5-23  gram.  Na2O  per  diem.  The  ratio 
K2O  :  Na2O  generally  equals  i  :  1-54. 

During  starvation  this  can  rise  as  high  as  3:1,  owing 
to  the  excretion  of  the  potassium  of  the  tissues,  sodium 
being  found  in  a  much  smaller  amount  than  potassium. 
The  same  is  found  in  all  wasting  diseases. 

Calcium  and  Magnesium  are  mainly  excreted  by  the 
bowel.  The  amounts  in  urine  are  0-33  to  0-6  gram.  CaO 
and  0-16  to  0-24  gram,  of  MgO. 

The  amounts  of  these  alkaline  earths  in  the  urine  are 
increased  by  the  administration  of  organic  acids,  or  in 
conditions  such  as  diabetes  in  which  the  formation  of  such 
acids  is  increased. 

Iron  also  is  mainly  excreted  by  the  bowel.  It  is  found 
in  human  urine  only  in  organic  combination,  and  then 
only  to  the  extent  of  0-5  to  10  milligrams  per  diem. 


CH.  XII.]  CHLORIDES   AND    SULPHATES.  281 

Anions. 

Chlorides  form  the  chief  part  of  the  anions  of  the 
urine.  The  amount  excreted  is  often  calculated  as  if  it  all 
existed  as  NaCl,  though  the  amount  of  sodium  in  the  urine 
is  normally  not  sufficient  to  combine  with  all  the  chlorine. 
The  amount  in  the  urine  depends  largely  on  the  amount  in 
the  food,  but  since  an  important  function  of  the  kidney  is 
to  maintain  a  constant  osmotic  pressure  of  the  tissue 
fluids,  mainly  by  variations  in  the  amount  of  NaCl  excreted, 
it  follows  that  anything  tending  to  cause  a  change  in  the 
osmotic  equilibrium  in  the  body  is  liable  to  alter  the 
excretion  of  chlorides  in  the  urine. 

Thus  during  starvation  and  during  the  formation  of 
exudates  in  pneumonia  the  chlorides  may  disappear  from 
urine.  The  amount  of  Cl  excreted  per  diem  is  about  7 
grams.  Reckoned  as  NaCl  it  is  12  grams. 

For  the  method  of  estimation  see  p.  352. 

Sulphates.  Only  a  small  portion  of  the  sulphate 
excreted  in  the  urine  is  taken  in  as  such  with  the  food. 
The  greater  portion  is  derived  from  the  oxidation  of  sulphur 
containing  substances,  chiefly  proteins.  The  amount 
of  sulphates  is  thus  a  rough  measure  of  the  total  amount 

N  ^ 

of  protein  metabolised,  the  ratio  — — -  being  usually  - 

SO3  i 

Sulphates  are  excreted  very  rapidly  after  a  protein 
meal,  reaching  a  maximum  about  the  third  hour.  This 
seems  to  indicate  that  cystine,  the  sulphur  complex  of 
proteins,  is  split  off  and  absorbed  very  early  in  the  digestion 
of  proteins. 

Ethereal  Sulphates  are  esters  formed  by  the  union  of 
sulphuric  acid  with  phenols. 

O    OH  O    O.C6H5 

\/  V/ 

S          +     HO.C6H5    —     =>        g  +     H20 

<f\  Xx\ 

O    OH  O    OH 

Sulphuric  acid-  Phenol.  Phenyl  sulphuric  acid. 


282  URINE.  [CH.  XII 

The  proportion  of  the  sulphur  that  is  present  as  ethereal 
sulphate  varies  considerably  Folin  has  shewn  that  in 
starvation  and  on  diets  relatively  deficient  in  proteins  the 
proportion  increases,  as  does  that  of  the  "neutral"  sulphur. 
There  is  also  a  marked  increase  after  the  administration  of 
certain  phenolic  substances,  or  when  such  compounds  are 
formed  in  the  body  by  bacterial  decomposition,  as  in 
intestinal  obstruction  and  severe  constipation.  In  such 
cases  the  phenols  found  conjugated  with  sulphuric  acid  are 

C6H5.OH  ...............  phenol 


......  p-cresol 


fr°m  tyrosine' 


C9H6N.OH  ............  indoxyl,  formed  from  tryptophane. 

These  bodies  are  poisonous.  They  unite  with  sulphuric 
acid,  probably  in  the  liver,  to  form  the  innocuous  ethereal 
sulphates. 

The  ethereal  sulphates  form  soluble  barium  and 
benzidine  salts,  and  can  be  separated  from  the  inorganic 
sulphates  by  treatment  with  barium  chloride  or  benzidine 
hydrochloride  and  filtering.  They  are  hydrolysed  to  the 
phenol  and  sulphuric  acid  by  boiling  with  hydrochloric  acid. 

"Neutral"  Sulphur.  In  urine  there  is  always  present  a 
certain  amount  of  sulphur  in  a  form  less  oxidised  than 
that  of  a  sulphate.  The  exact  nature  of  the  compounds 
in  urine  containing  sulphur  in  this  form  is  not  yet  clear. 

It  is  probable  that  the  amount  of  "neutral"  sulphur 
in  the  urine  is  independent  of  the  total  amount  of  sulphur 
excreted.  It  probably  varies  with  the  amount  of  tissue 
protein  metabolised,  so  that  its  determination  is  often  of 
considerable  interest. 

For  the  percentages  of  sulphur  excreted  in  the  three 
forms  under  different  metabolic  conditions  see  page  270. 

For  the  methods  of  determination  of  the  sulphur  see 
pages  355—358. 

Phosphates.  The  phosphates  of  the  urine  are  present 
on  the  one  hand  as  salts  of  the  alkali  metals  and  of 


CH.  XII. 1  PHOSPHATES.  283 

ammonium  ;  on  the  other,  as  salts  of  the  alkaline  earths, 
calcium  and  magnesium.  About  3-9  grams,  of  P2O5  are 
excreted  per  diem  in  the  urine.  Phosphoric  acid  forms 
three  series  of  salts.  The  formulae  for  that  of  sodium  and 
calcium  are 

Normal  phosphate,  Na3PO4  :  Ca^POJg. 

Mono-hydrogen  phosphate,  Na2HPO4  :  CaH(PO4). 

Di-hydrogen  phosphate,  NaH2PO4        :  CaH4(PO4)2. 

The  three  sodium  salts  and  CaH4(PO4)2  are  soluble  in 
water  :  the  other  two  calcium  salts  are  insoluble.  The 
normal  and  mono-hydrogen  phosphates  are  alkaline  in 
reaction  to  litmus  :  the  di-hydrogen  phosphates  are  acid. 

The  phosphates  of  the  urine  are  derived  partly  from 
the  inorganic  phosphates  of  the  food,  partly  from  the 
oxidation  of  phosphorus-containing  substances  of  the 
food  and  tissues,  such  as  nucleo-proteins,  lecithins  and 
phospho-proteins,  and  partly  also  from  the  phosphates  of 
bone.  The  exact  share  played  by  these  various  compounds 
in  forming  the  urinary  phosphates  is  difficult  to  deter- 
mine owing  to  the  fact  that  a  proportion  of  the  phosphates, 
varying  between  12  and  50  per  cent.,  are  excreted  by  the 
bowel.  In  this  connection  it  may  be  noted  that  alkaline 
phosphates  of  the  food  are  more  likely  to  be  excreted  in 
the  urine  than  are  earthy  phosphates. 

The  excretion  of  varying  amounts  of  phosphates  by 
the  kidney  is  one  of  the  methods  by  means  of  which  the 
reaction  of  the  body  fluids  is  maintained  in  equilibrium. 
An  increased  excretion  is  always  seen  in  cases  of  acid 
poisoning  and  in  the  acidosis  associated  with  diabetes. 

As  soon  as  the  urine  shews  a  certain  grade  of  alka- 
linity, precipitation  of  earthy  phosphates  takes  place. 
This  is  sometimes  known  as  phosphaturia,  but  it  is  not 
necessarily  associated  with  an  increase  of  phosphates  in 
the  urine.  In  the  phosphaturia  of  juveniles  it  is  probable 
that  there  is  an  excessive  amount  of  calcium  in  the  urine, 
due  to  a  defective  excretion  of  the  large  intestine. 


284  URINE.  [CH.  XII. 

A  certain  amount  of  phosphorus  is  found  in  the  urine 
in  an  organic  form,  not  as  a  phosphate.  It  may  be  present 
as  glycero-phosphoric  acid.  The  average  daily  amount  is 
about  50  mgms. 

For  method  of  estimation  see  Ex.  414. 

327.  Test  for  chlorides  by  adding  to  about  3  cc.  of  urine  a 
few  drops  of  pure  nitric  acid  and  3  cc.  of  a  3  per  cent,  solution  of 
silver  nitrate.     An  abundant  curdy  precipitate  of  silver  chloride 
appears  at  once.     If  the  chlorides  are  less  in  quantity,  the  solution 
merely  becomes  milky  or  opalescent. 

NOTE. — If  nitric  acid  is  not  added,  urates  might  be  precipitated  by  silver 
nitrate,  especially  if  the  urine  be  ammoniacal. 

328.  To  a  test-tube  nearly  full  of  urine  add  a  little  strong 
ammonia  and  boil.     A  white  flaky  precipitate  of  the  phosphates 
of  calcium  and  magnesium  is  formed.     Filter  off  the  precipitate, 
wash  with  water,  and  dissolve  in  5  cc.  of  dilute  acetic  acid.     Divide 
the  solution  into  two  parts.     To  one  part  add  a  solution  of  potassium 
oxalate.     A  white  precipitate  is  produced,  showing  the  presence  of 
calcium  in  the  urine. 

329.  To  the  other  portion  of  the  solution  add  an  equal  bulk 
of  strong  nitric  acid  and  about  5  cc.  of  ammonium  molybdate. 
Boil :    a  yellow  crystalline  precipitate  is  produced,  showing  the 
presence  of  phosphates. 

NOTE. — Neutral  urine  is  very  apt  to  yield  a  precipitate  of  earthy  phos- 
phates on  boiling,  owing  to  the  change  of  reaction  due  to  the  evolution  of 
CO2  (see  notes  to  Ex.  28). 

330.  To   demonstrate    the   presence  of    acid-phosphates  in 
urine.     Treat  5  cc.  of  urine  with  an  equal  volume  of  5  per  cent, 
solution  of  barium  chloride.     Filter  repeatedly  through  a  small 
filter  paper  till  the  filtrate  is  clear.     Treat  the  filtrate  with  a  little 
baryta   mixture   and   boil.     Filter;     dissolve   the   precipitate   in 
nitric  acid  and  boil  the  solution  obtained  with  ammonium  molybdate. 
The  yellow  precipitate  shows  the  presence  in  the  urine  of  acid 
phosphates,  such  as  NaH2PO4. 


CH.  XII.]  ETHEREAL  SULPHATES.  285 

NOTE. — Any  alkaline  phosphate,  Na2HPO4,  present  in  the  urine  is  precipi- 
tated by  BaClj  as  BaHPO4.  The  acid  phosphates  remain  in  solution  as 
Ba(H2PO4)2.  On  the  addition  of  the  alkaline  baryta  mixture,  the  acid  phos- 
phate is  converted  into  the  insoluble  alkaline  phosphates  of  barium.  li  no 
precipitate  is  produced  when  the  baryta-mixture  is  added,  there  are  no  acid 
phosphates  present  in  the  sample  of  urine. 

Since  the  acidity  of  a  sample  of  urine  varies  almost  directly  with  the 
amount  of  acid  phosphates  present,  as  determined  by  the  above  method,  it  is 
generally  held  that  the  acidity  of  urine  is  mainly  due  to  the  presence  of  these 
acid  phosphates. 

331.  Treat  10  cc.  of  urine  with  a  few  drops  of  strong  hydro- 
chloric acid,  and  about  3  cc.  of  a  solution  of  barium  chloride.  A 
precipitate  of  barium  sulphate  is  produced  as  an  opaque  milkiness. 
If  the  precipitate  is  thick  the  sulphates  are  in  excess.  (The  hydro- 
chloric acid  is  added  to  prevent  the  precipitation  of  phosphates.) 


332.    To   demonstrate  the   presence  of   ethereal   sulphates. 

To  urine  add  an  equal  bulk  of  baryta  mixture  (two  parts  of  baryta 
water  to  one  part  of  a  10  per  cent,  solution  of  barium  nitrate).  A 
precipitate  is  formed  consisting  of  the  phosphates  and  the  ordinary 
inorganic  sulphates.  Filter  till  quite  clear.  To  the  nitrate  add  a 
third  of  its  volume  of  strong  hydrochloric  acid,  boil  in  a  beaker  for 
five  minutes,  and  allow  to  stand.  A  faint  white  cloud  of  barium 
sulphate  is  formed,  indicating  the  presence  of  ethereal  sulphates  in 
the  urine. 

NOTES. — i.     The  ethereal  sulphates  form  soluble  barium^salts,  but  are 
hydrolysed  to  sulphuric  acid  by  heating  with  an  acid. 

c6H5  -  o  _ 

_^SO2  +  H2O=C6H6.OH  +  HaSo4. 

HO  Phenol. 

Phenyl-sulphuric  acid. 

The  sulphuric  acid  thus  formed  is  converted  into  bariiim  sulphate  by  the 
excess  of  barium  present. 

2.  The  solution  becomes  very  dark  in  colour  on  boiling  with  the  strong 
acid,  owing  to  the  action  of  the  latter  on  the  aromatic  chromogenic  substances 
in  the  urine. 

3.  Ethereal  sulphates  can  be  prepared  as  follows:  warm  10  drops  of 
absolute  alcohol  with  5  drops  of  concentrated  sulphuric  acid  in  a  test-tube. 
Cool  and  make  alkaline  with  5  per  cent.  soda.     Add  10  per  cent,  barium 
chloride  as  long  as  a  precipitate  continues  to  be  formed.     Boil  and  filter. 
The  filtrate  con  tarns  barium  ethyl  sulphate.    Add  one-half  volume  of  concen- 
trated  hydrochloric   acid    and  boi!.     A   precipitate  of   barium   sulphate   is 
formed. 


286  URINE.  [CH.  XII. 

E.    Urea. 

Urea  is  the  compound  in  which  the  greater  part  of 
the  nitrogen  is  normally  excreted  in  man.  The  percentage 
of  the  urinary  nitrogen  in  the  form  of  urea  varies.  Normally 
it  is  about  86  per  cent.,  but  in  starvation,  or  on  a  diet 
deficient  in  proteins,  it  is  only  about  60  per  cent.  It  is 
also  low  in  cases  of  diabetes  accompanied  by  acidosis 
(owing  to  the  relatively  high  percentage  of  ammonia),  and 
also  in  certain  cases  of  hepatic  disorder,  notably  acute 
yellow  atrophy  of  the  liver,  owing  to  the  non-formation 
of  urea  by  the  disordered  liver,  its  seat  of  formation  in  the 
body. 

The  total  amount  excreted  per  diem  by  a  normal  man 
on  an  average  diet  containing  100  grams,  of  protein  is 
30  grams. 

Urea  is  also  known  as  carbamide,  since  it  is  the  diamide 
of  carbonic  acid. 

o-c— -OH  o   r— NH* 

-OH  -NH^ 

Carbonic  acid.  Urea. 

Urea  crystallises  in  water-free,  colourless,  long  needles, 
or  in  four-sided  prisms  of  the  rhombic  system,  which  melt 
and  decompose  at  130°  —  132°  C. 

It  is  soluble  in  all  proportions  in  hot  water,  and  to 
the  extent  i  :  i  in  cold  water.  In  cold  alcohol  it  is  soluble 
to  the  extent  of  i  :  5.  It  is  also  soluble  in  acetone.  In- 
soluble in  pure  ether  and  chloroform.  The  solutions  are 
neutral  in  reaction. 

It  forms  crystalline  compounds  with  acids.  The  two 
most  important  are  urea  nitrate  CH4N2O.HNO3,  insoluble 
in  strong  nitric  acid,  and  urea  oxalate  (CH4N2O)2,  C2H2O4, 
insoluble  in  oxalic  acid. 

It  forms  compounds  with  the  salts  of  the  heavy  metals, 
especially  with  mercuric  nitrate  (see  below,  Ex.  341). 

With  reducing  sugars  relatively  stable  compounds 
are  formed,  called  ureides.  They  are  of  importance  in 
connection  with  the  estimation  of  urea  in  diabetic  urine. 


CH.  XII.]  UREA.  287 

On  heating  dry  urea  to  140°  C.,  ammonia  is  evolved 
and  biuret  formed. 

NHa 

NH9 

\  CO 

NH3 

NH        +    NH, 
NH,  I 

/  CO 

CO  \ 

NH2 
NH, 

On  heating  beyond  140°  C.,  cyanuric  acid  and  ammonia 
are  formed.     Cyanuric  acid  is  QH-jNgOg. 

N 
HO— C    C— OH 


I     II 
N    N 


V 

C— OH 

Solutions  of  urea  are  decomposed  by  boiling  alkalies 
into  CO2  and  NH3.  They  are  also  similarly  decomposed 
by  heating  for  several  hours  at  150°  C.  with  acids.  This 
decomposition  is  readily  effected  by  the  addition  of 
magnesium  chloride,  zinc  sulphate  or  potassium  acetate 
to  the  solution  for  the  purpose  of  raising  the  boiling 
point. 

Bacteria,  as  micrococcus  ureae,  decompose  urea  into 
CO2  and  NH3.  This  accounts  for  normal  urine  rapidly 
becoming  ammoniacal  on  standing  in  the  air. 

Urea  is  decomposed  by  the  enzyme  urease  into  am- 
monium carbonate.  The  enzyme  is  found  in  the  Soya  bean 
and  in  other  plants.  It  does  not  act  on  any  other  com- 
pound, not  even  on  the  substituted  ureas.  It  is  therefore 
used  both  for  the  detection  (Ex.  343)  and  also  for  the 
estimation  (Ex.  401)  of  urea. 


288  URINE.  [CH.  xn. 

Nitrous  acid  decomposes  urea  as  follows  :— 
CO(NH2)2  +  2HNO2  =  2N2  +  CO2  +  sH2O. 

Hypobromites  effect  a  similar  decomposition. 
CO(NH2)2  +  3NaBrO  =  sNaBr  +  CO2  +  N2  +  2H2O 

Sodium 
hypobromite. 

According  to  Werner*  the  reactions  of  urea  are  better 
understood  if  it  be  supposed  that  it  exists  in  two  tauto- 
meric  modifications,  the  equilibrium  between  them 
depending  on  the  reaction 

/OH 

HN  =  C^  HN  =  C'x 

\NH2  NH3 

A.  B. 

The  A  form  exists  in  strongly  acid  solutions.  It  is 
decomposed  by  nitrous  acid,  like  all  compounds  with  the 
NH2  group.  The  B  form  exists  in  neutral  or  alkaline 
solutions  and  is  not  decomposed  by  nitrous  acid.  >For  the 
convincing  evidence  on  which  this  view  is  based  the 
original  papers  should  be  consulted. 

333.  To  a  watch-glass  half  full  of  distilled  water  add  as  much 
solid  urea  as  will  lie  on  a  sixpenny-piece.     Note  the  solubility  of  urea 
in  water. 

334.  Place  a  drop  of  the  urea  solution  on  a  slide,  add  a  single 
drop  of  a  saturated  solution  of  oxalic  acid,  mix  by  stirring  with  a 
needle  or  fine  glass  rod,  cover  with  a  slip  and  examine  the  crystals 
of  oxalate  of  urea  that  separate  out.     They  vary  considerably, 
containing  long,  thin,  flat  crystals,  often  in  bundles  and  rhombic 
prisms.     Draw  the  crystals. 

*  Journal  Chem.  Soc.y  cix.,,p.  1120. 


CH.  XII.]  UREA.  289 

335.  Dilute  the  urea  solution  with  twice  its  volume  of  water. 
Place  a  drop  on  a  slide,  add  a  drop  of  pure  nitric  acid,  cover  with  a 
slip,  and  examine  the  crystals  of  urea  nitrate  that  separate  out. 
They  form  octahedral,  lozenge-shaped,  or  hexagonal  plates,  often 
striated  and  imbricated.     Draw  the  crystals. 

336.  Powder  two  or  three  crystals  of  urea  in  a  watch-glass  : 
rub  with  a  small  amount  of  acetone  and  warm  gently  on  a  water 
bath.     The  urea  dissolves.     Allow  most  of  the  acetone  to  evaporate 
away,  and  then  place  a  drop  of  the  remaining  solution  on  a  watch- 
glass.     Urea  crystallises  out  as  the  acetone  passes  off.     Draw  the 
crystals. 

337.  Repeat  the  above  exercise,  using  strong  alcohol  instead 
of  acetone.     Draw  the  crystals  of  urea,  which  are  usually  very 
irregular. 

338.  Dilute  the  remainder  of  the  aqueous  solution  left  from 
Ex.  335  with  an  equal  quantity  of  water,  and  to  a  portion  of  this  in 
a  test-tube  add  some  yellow  nitric  acid  (or  nitric  acid  to  which  a 
little  potassium  nitrite  has  been  added).     An  effervescence  and 
evolution  of  gas  take  place. 

CO(NH2)2  +  2HN02  =  C02  +  2N2  +  3H2O. 

NOTE.  —  All  compounds  containing  the  amino  group  (NHj)  react  in  a 
similar  manner  when  treated  with  nitrous  acid  (see  Ex.  81).  The  decompo- 
sition of  urea  with  nitric  acid  is  relatively  very  slow. 

339.  To  another  portion  of  the  solution  add  sodium  hypo- 
bromite.     A  marked  effervescence  and  evolution  of  gas  take  place. 

CO(NH2)2  +  3NaBrO  +  2NaHO 

=  3NaBr  +  N^CC^  +  3H20  +  N2. 


340.  To  a  few  cc.  of  saturated  ammonium  sulphate  add 
sodium  hypobromite.  A  marked  effervescence  and  evolution  of  gas 
take  place. 

(NH4)2SO4  +  3NaBrO  +  2NaHO 

=  Na2SO4  +  5H2O  +  aNaBr  +  N2. 


NOTES.  —  i.  All  ammonium  salts  and  all  compounds  with  the  amino 
group  give  off  nitrogen  when  treated  with  an  alkaline  solution  of  sodium  hypo- 
bromite. 


290  URINE.  [CH.  XII. 

2.  The  sodium  hypobromite  is  prepared  as  follows  :  dissolve  100  grams. 
of  caustic  soda  in  250  cc.  of  water.     Cool,  and  slowly  add  25  cc.  of  bromine, 
cooling  under  the  tap  as  the  bromine  is  added.     The  reaction  is  as  follows  : 

2  NaHO  +  Br2  =  NaBrO  +  NaBr  +  H2O. 

It  must  be  freshly  prepared  before  use  as  it  undergoes  the  following 
decomposition  : 

3  NaBrO  =  2  NaBr  +  NaBrO3. 

3.  As  a  test  for  urea  the  reaction  with  hypobromite  is  only  useful  in  a 
negative  sense;    that  is  to  say,  if  an  effervescence  is  not  obtained  urea  is 
absent,  but  if  an  effervescence  is  obtained  it  does  not  necessarily  follow  that 
urea  is  present. 

341.  To  some  of  the  urea  solution  add  a  solution  of  mercuric 
nitrate.     A  white  precipitate  of  mercuric  oxide  combined  with  urea 
and  mercuric  nitrate  takes  place.     To  the  mixture  thus  obtained 
add  a  saturated  solution  of  sodium  chloride,  drop  by  drop.     The 
precipitate  dissolves,  to  reappear  on  a  further  addition  of  mercuric 
nitrate. 

NOTES. — i.  The  precipitate  consists  of  urea  and  mercuric  nitrate  and 
one,  two  or  three  molecules  of  mercuric  oxide,  depending  on  the  concentration 
of  the  two  solutions. 

2.  The  solubility  in  NaCl  is  due  to  the  formation  of  mercuric  chloride, 
which  is  only  very  feebly  ionised  in  neutral  solutions. 

342.  Treat  a  solution  of  urea  with  Millon's  reagent,  and  heat. 
A  white  precipitate  is  formed,  owing  to  the  presence  of  mercuric 
nitrate  in  the  reagent.     There  is  also  an  evolution  of  gas  due  to  the 
action  of  the  nitrous  acid  on  the  urea. 

343-    Specific  urease  test  for  urea.     To  4  or  5   cc.   of  a 

dilute  solution  of  urea  add  4  or  5  drops  of  phenol  red.  The  colour 
obtained  is  generally  slightly  pinkish.  Add  traces  of  very  dilute 
acetic  acid  by  means  of  a  glass  rod  until  the  reaction  is  very  faintly 
acid  to  the  indicator.  Warm  to  about  45°  C.  Add  a  large  "knife 
point  "  of  finely  ground  Soya  bean  meal,  shake  and  keep  the  solution 
warm.  The  colour  changes  to  a  reddish  purple,  owing  to  the 
enzyme  converting  neutral  urea  to  alkaline  ammonium  carbonate. 

NOTES. — i.  In  applying  the  test  it  is  important  to  see  that  the  reaction 
is  only  faintly  acid  to  the  indicator.  For  if  a  considerable  amount  of  acid 
and  only  a  small  amount  of  urea  be  present,  the  amount  of  ammonium  carbon- 
ate formed  may  not  be  sufficient  to  bring  the  reaction  to  the  point  where  a 
pink  colour  is  given  with  the  indicator. 

2.  Proteins  only  interfere  by  acting'as  buffers.  It  is  not  usually  neces- 
sary to  remove  them. 


CH.  XII.]  UREA.  291 

3.  The  test  will  not  succeed  in  the  presence  of  the  salts  of  the  heavy 
metals,  which  inhibit  the  action  of  the  enzyme.  A  high  concentration  of 
buffer  salts,  such  as  phosphates  or  acetates,  decreases  the  delicacy  of  the  test, 
by  preventing  considerable  changes  in  hydrogen-ion  concentration. 

344.  To  about  4  cc.  of  a  I  per  cent,  solution  of  urea  add  about 
2  cc.  of  strong  soda,  mix  and  divide  into  two  portions,  A  and  B. 
Boil  B  for  3  to  5  minutes,  adding  a  little  water  from  time  to  time  to 
replace  that  lost  by  evaporation.     Cool  under  the  tap.     Add  phenol 
red  to  each  and  neutralise  by  the  addition  of  hydrochloric  acid,  using 
concentrated  acid  at  first  and  finish  by  dilute  hydrochloric.     Apply 
the  urease  test  as  described  in  the  previous  exercise  to  the  two 
solutions.     A  gives  a  strong  test,  whilst  B  gives  none,  or  only  a 
slight  one,  owing  to  the  destruction  of  the  urea  by  boiling  alkali. 

345.  Place  a  little  urea  in  a  dry  test-tube.     Heat  carefully 
over  a  flame,  keeping  the  upper  part  of  the  tube  cool.    The  urea 
melts  and  evolves  ammonia,  whilst  a  white  sublimate  condenses  on 
the  cooler  parts  of  the  tube.     Cool  the  tube,  add  a  little  water  and 
shake.     Pour  the  solution  into  another  tube  and  treat  it  with  an 
equal  bulk  of  sodium  hydroxide  and  a  drop  of  copper  sulphate.     A 
pink  colour  is  produced,  due  to  the  biuret  formed  from  the  urea. 

346.  Repeat  the  experiment,  but  heat  more  strongly  till  the 
melt  solidifies  and  becomes  opaque.     Cool,  add  two  or  three  cc. 
of  water,  boil  and  filter  whilst  still  hot.     Divide  the  solution  into 
two  portions,  A  and  B.     To  A  add  a  few  drops  of  a  solution  of 
barium  chloride  and  a  single  drop  of  diluted  ammonia.     A  white 
mass  of  barium  cyanurate  is  formed  on  cooling. 

To  B  add  some  ammoniacal  copper  sulphate  solution  and  boil. 
On  cooling  an  amethyst  precipitate  of  copper  ammonium  cyanurate 
is  deposited. 

NOTE. — Preparation  of  ammoniacal  copper  sulphate,  i  per  cent,  copper 
sulphate  is  treated  with  very  dilute  ammonia  till  the  precipitate  that  first  forms 
just  redissolves. 

347.  Isolation  of  urea  from  urine.    Evaporate  about  30  cc. 
of  urine  to  complete  dryness,  finishing  the  evaporation  on  the  water 
bath  (to  prevent  the  destruction  of  the  urea) .    Turn  out  the  flame 
and  rub  the  residue  with  about  10  cc.  of  acetone  till  it  is  boiling. 
Allow  the  acetone  to  boil,  stirring  all  the  time,  till  about  half  of  it 


2Q2  URINE.  [CH.  XII. 

has  evaporated  away.  Pour  off  the  acetone  into  a  dry  watch  glass 
and  allow  it  to  cool.  Crystals  of  urea  separate  out  as  silky  needles. 
Demonstrate  that  they  are  urea  crystals  by  evaporating  to  dryness, 
taking  up  in  a  small  amount  of  water  and  applying  the  urease  test 
(Ex.  343). 

F.    Uric  Acid. 

Uric  Aci£,  C5H4N4O3,  is  2-6-  8-tri-oxy-p  urine. 
NH-CO 

CO       C  -  NH^ 

I!  CO 

NH  -  C  - 


Its  relationship  to  certain  of  the  other  purines  is 
indicated  on  page  63. 

When  pure  it  crystallises  in  microscopic  rhombic 
plates,  but  when  impure  it  assumes  a  variety  of  forms, 
such  as  whetstones,  dumb-bells,  sheaves,  rosettes,  butchers1 
trays,  etc. 

It  dissolves  to  the  extent  of  i  part  in  16,000  parts  of 
cold  water  and  i  ,600  parts  of  hot  water.  It  dissolves  in 
alkalies,  and  the  alkali  salts  of  carbonic,  phosphoric,  boric, 
lactic  and  acetic  acids,  but  not  in  the  ammonium  salts  of 
these  acids.  It  dissolves  in  warm  concentrated  sulphuric 
acid  to  form  a  sulphate,  which  is  decomposed  by  the  addi- 
tion of  water. 

It  is  precipitated  by  phosphotungstic  acid  in  the 
presence  of  hydrochloric  acid,  slowly  by  lead  acetate,  and 
completely  by  picric  acid,  mercuric  chloride  and  ammonia- 
cal  silver  nitrate. 

By  oxidation,  allantoin,  alloxan,  parabanic  acid  and 
urea  are  formed,  depending  on  the  reaction  and  the  reagent 
employed. 

NHa  NH  -  CO  NH  -  CO 

CO      CO  -  NH^  CO      CO  CO 

NH  -  CH  -  NH-  NH  -  CO  NH  -  CO 

Allautoin.  Alloxan.  Parabanic  acid. 


CH.  XII.]  URIC   ACID.  293 

Although  the  aqueous  solutions  of  uric  acid  react 
neutral,  it  behaves  like  a  disbasic  acid  C5H2N4O3.H2  and 
can  form  two  series  of  salts,  C5H2N4O3.Na2  (neutral,  normal, 
or  di-sodium  urate)  and  C5H2N4O3.HNa  (biurate,  acid  urate 
or  mono-sodium  urate).  It  is  also  possible  that  there  is  a 
third  form  of  salt,  C5H2N4O3.HNa.C5H4N4O3  (quadriurate 
or  hemi-sodium  urate),  though  this  may  be  merely  a  mixture 
of  its  two  constituents.  The  di-sodium  salts  are  more 
soluble  than  the  mono-sodium,  but  are  only  stable  in 
markedly  alkaline  solutions.  In  the  blood  and  urine 
urates  exist  as  mono-sodium  salts,  which  react  neutral. 

It  is  interesting  to  note  that  there  are  two  modifica- 
tions of  the  mono-sodium  salt,  called  the  a-  and  /3-form. 
The  a-form  is  more  soluble  than  the  /3-form,  but  is  un- 
stable, and  slowly  passes  over  into  the  other  form.  They 
are  probably  the  salts  of  the  two  tautomeric  modifications 
of  uric  acid  described  by  Fischer  : 

NH  -  CO  N  =  C.OH 

CO       C-NH^  HO.C      C-NH^ 

>         ||       ||  CO 


-  C  -  NH^  N  -  C  - 

Lactam  modification  forming  Lactim  modification  forming 

unstable  a-urate.  stable  /?-urate. 

It  is  of  great  interest  to  observe  that  in  gout  the 
amount  of  urate  in  solution  in  the  blood  is  in  excess  of  the 
amount  of  the  /3-  urate  that  can  be  held  by  normal  blood. 
So  that  in  gout  it  must  be  present  at  least,  partly,  in  the 
unstable  a-form.  The  deposition  of  urates  in  the  tissues 
during  an  acute  attack  may  be  due  to  the  conversion  of  the 
unstable  a-  into  the  stable,  less  soluble  ^-modification. 

Urates  are  completely  precipitated  as  amorphous 
ammonium  urate  by  saturation  with  ammonium  chloride. 

They  exert  a  reducing  reaction  on  Fehling's  solution 
and  towards  alkaline  silver  solutions,  this  being  the  basis 
of  Schiff's  test. 

They  yield  a  characteristic  colour  reaction  when 
evaporated  with  nitric  acid,  the  so-called  murexide  test. 


294  URINE.  [CH.  XII. 

Uric  acid  occurs  to  the  extent  of  about  0-7  gram,  in  the 
24  hours'  urine,  but  the  amount  excreted  varies  with  the 
diet  and  the  individual. 

From  its  close  chemical  relationship  to  the  purine 
bases  formed  by  the  hydrolysis  of  the  nucleins  of  the  food 
and  tissues  (see  p.  63),  the  view  is  commonly  held  that 
uric  acid  has  its  origin  in  the  cellular  organs  of  the  body 
from  the  oxidation  of  such  substances.  Thus  we  can  have 
uric  acid  arising  exogenously  from  the  free  or  combined 
purines  of  the  food  and  also  endogenously  from  those  of 
the  tissues.  This  view  is  apparently  supported  by  the 
fact  that  the  administration  of  foods  rich  in  nucleoproteins, 
as  sweetbreads,  or  of  certain  of  the  pure  purine  bases, 
does  cause  an  increased  excretion  of  uric  acid. 

It  is  possible  that  a  certain  proportion  of  the  uric  acid 
formed  in  the  body  is  destroyed  by  the  liver,  so  that  the 
amount  excreted  is  a  balance  between  that  formed  and  that 
destroyed. 

In  gout,  in  which  there  is  a  deposition  of  uric  acid  in 
the  tissues,  the  excretion  is  decreased  before  an  acute 
attack,  is  increased  during  the  attack,  and  then  falls  again. 
In  this  condition  there  is  a  recognisable  amount  of  uric 
acid  in  the  blood  (see  above).  For  the  method  of  estima- 
tion in  urine  see  Ex.  406. 

348.  Treat  a  small  amount  of  uric  acid  with  10  cc.  of  2  per 
cent,  sodium  carbonate.     Heat  nearly  to  boiling  and  cool.     Note 
that  a  considerable  portion  of  the  uric  acid  has  dissolved  in  the  form 
of  a  urate. 

349.  Filter  the  solution  and  treat  a  portion  with  a  drop  or  two 
of  strong  hydrochloric  acid  and  shake.     A  white  crystalline  pre- 
cipitate of  uric  acid  separates  out,  showing  that  uric  acid  is  very 
insoluble  in  water.     Allow  the  crystals  to  settle,  remove  a  few  by 
means  of  a  pipette,   and  examine  them  microscopically.    They 
usually  form  rhombic  plates.     Draw  the  crystals. 

NOTE. — If  the  solution  is  very  strong,  the  uric  acid  may  separate  out  in  an 
amorphous  form.  Should  this  be  the  case,  make  the  solution  alkaline  and  heat 
to  dissolve.  Whilst  still  hot  add  some  I1C1  and  allow  the  tube  to  cool  slowly. 


CH.  XII.]  URIC    ACID.  295 

Uric  acid  can  assume  a  great  variety  of  crystalline  forms,  resembling 
dumb-bells,  whetstones,  butcher-trays,  stars,  and  sheaves. 

350.  To  another  portion  of  the  solution  add  two  drops  of 
ammonia  and  saturate  with  ammonium  chloride.     A  white  amor- 
phous precipitate  of  ammonium  urate  is  formed. 

NOTE. — This  is  the  basis  of  Hopkins'  original  method  for  the  estimation  of 
urates  in  urine.  It  is  an  important  reaction  for  separating  urates  from  physio- 
logical fluids,  such  as  urine  (see  Ex.  359),  since  no  other  organic  substance, 
likely  to  be  met  with  in  physiological  analysis,  is  precipitated  by  saturation 
with  ammonium  chloride.  The  murexide  reaction  can  be  applied  to  the 
precipitate  obtained. 

351.  Treat  a  little  uric  acid  with  a  little  strong  sulphuric  acid  : 
it  dissolves.     Pour  the  solution  into  water:  the  uric  acid  may 
separate  out. 

352.  Murexide  test.    Treat  a  little  uric  acid  in  a  porcelain 
dish  with  two  or  three  drops  of  strong  nitric  acid.     Heat  on  the 
water  bath  till  every  trace  of  nitric  acid  and  water  has  been  re- 
moved.    A  reddish  deposit   remains.    Treat   this  with  a  dilute 
solution  of  ammonia  (five  drops  of  ammonia  to  about  a  test-tube 
full  of  water).     The  residue  turns  reddish- violet  in  colour.     Add 
a  little  caustic  soda.    The  colour  turns  to  a  blue- violet. 

NOTES. — i.  This  important  test  needs  a  certain  amount  of  care.  The 
heating  must  be  performed  on  the  water-bath,  and  should  be  continued  as 
long  as  is  necessary  to  ensure  the  complete  removal  of  every  trace  of  nitric  acid. 

2.  Xan thine  and  guanine  give  a  yellow  substance  (nitro-xanthine)  when 
treated  with  nitric  acid.     On  evaporation  the  colour  goes  to  a  violet  shade, 
which  turns  yellow  with  dilute  ammonia.     Adenine  and  hypoxanthine  give  no 
colour  reaction. 

3.  The  chemistry  of  the  reaction  is  as  follows :  From  uric  acid  arises 
by  oxidation  dialuric  acid  and  alloxan.     They  condense  together  to  form 
alloxantin.     By  the  action  of  ammonia  on  alloxantin,  purpuric  acid  is  formed. 
Murexide  is  ammonium  purpurate, 

HN-CO  HN-CO  HN-CO  OC-NH 

I       I      H                |i                                     |       I        OH  |        | 

OC     C-^          +   OC     CO  =  OC     C^i C     CO 

!     I  XOH         ||  II"   HO^-  |      I 

HN-CO  HN-CO  HN-CO  OC-NH 

Dialuric  acid  Alloxan.  Alloxantin. 

__  NH       OC-NH 

I       I        "J> 
Alloxantin  +  NH8  • 

HN-CO  OC-NH 

Purpuric  acid. 


2Q6  URINE.  [CH.  XII. 

353.  Schiff's  test.    Treat  a  very  small  amount  of  uric  acid 
with  a  few  cc.  of  sodium  carbonate.       Pour  the  solution  on  to 
filter  paper  moistened  with  silver  nitrate.    A  black  stain  of  reduced 
silver  immediately  results. 

NOTE. — This  useful  test  cannot  be  applied  in  the  presence  of  chlorides.  It 
is  important  to  note  that  the  uric  acid  is  dissolved  in  sodium  carbonate,  not 
the  hydroxide,  as  the  latter  gives  a  precipitate  of  the  brown  silver  hydroxide, 
which  completely  obscures  the  reduction.  An  amount  of  sodium  carbonate  in 
excess  of  that  required  to  dissolve  the  uric  acid  must  be  added,  as  the  reduction 
only  takes  place  in  the  alkaline  condition. 

354.  Folin's  test.    To  a  very  small  pinch  of  uric  acid  in  a 
beaker  add  20  cc.  of  a  saturated  solution  of  sodium  carbonate. 
Stir  till  the  uric  acid  has  completely  dissolved,  add  I  cc.  of  Folin's 
uric  acid  reagent.    A  blue  colour  is  obtained. 

NOTES. — i.  Preparation  of  Folin's  solution.  100  grams,  of  pure  sodium 
tungstate,  102  cc.  of  pure  ortho- phosphoric  acid  (B.P.  66-3%)  and  750  cc.  of 
distilled  water  in  a  flask  fitted  with  a  reflux  condenser  are  boiled  for  2  hours. 
On  cooling  the  solution  is  diluted  to  i  litre. 

2.  The  solution  also  gives  a  blue  colour  with  polyphenols.  It  is  used  for 
the  microchemical  estimation  of  uric  acid  in  urine. 

355.  Dissolve  a  little  uric  acid  in  sodium  carbonate  by  boiling. 
Add  5  cc.  of  Fehling's  solution  and  boil  for  a  considerable  time. 
Note  the  peculiar  reduction  of  the  copper,  and  compare  it  with  the 
reduction  obtained  with  glucose. 

356.  Similarly  try  the  effect  of  uric  acid  on  Nylander's  (Ex. 
105)  and  Benedict's  (Ex.  100)  solutions.   A  reduction  is  not  obtained. 

357.  Dissolve  some  uric  acid  in  sodium  carbonate,  add  an 
excess  of  ammonia  and  treat  with  silver  nitrate.     A  white  amorphous 
precipitate  of  a  silver  compound  of  uric  acid  is  formed. 

NOTE. — Xanthine,  hypoxanthine  and  other  substances  in  urine  closely 
related  to  uric  acid  are  similarly  precipitated  by  ammoniacal  silver  nitrate. 

358.  A  solution  of  sodium  urate  and  urea  is  provided.   To 
prepare  crystals  of  uric  acid  and  of  urea. 

Heat  a  test-tube  nearly  full  of  the  solution  to  boiling  point  and 
add  strong  hydrochloric  acid  till  the  reaction  is  distinctly  acid. 
Allow  the  tube  to  cool  slowly ;  the  uric  acid  crystals  separate  out. 


CH.  XII.]  URIC    ACID.  297 

Cool  thoroughly  under  the  tap.  Filter  off  the  uric  acid.  Neutralise 
the  filtrate  with  sodium  carbonate  and  evaporate  to  dryness,  finish- 
ing the  process  on  the  water-bath,  to  prevent  the  conversion  of  the 
urea  to  biuret  (see  Ex.  345).  Extract  the  residue  with  strong 
alcohol  or  acetone.  The  alcohol  or  acetone  solution  is  carefully 
evaporated  to  dryness,  and  the  urea  crystallises  out. 

359.  To  demonstrate  the  presence  of  uric  acid  in  urine. 

Treat  50  cc.  of  urine  with  two  drops  of  ammonia  and  then  stir 
with  powdered  ammonium  chloride  till  the  solution  is  saturated. 
Allow  the  excess  of  ammonium  chloride  to  settle  for  15  sees.,  and 
pour  off  into  another  beaker.  Note  the  gelatinous  precipitate  of 
ammonium  urate.  Filter :  scrape  the  precipitate  off  the  paper  and 
transfer  it  to  an  evaporating  dish.  Add  three  or  four  drops  of  strong 
nitric  acid  and  place  the  dish  on  the  water-bath  till  a  pink,  dry 
residue  is  obtained.  Treat  this  with  a  little  dilute  ammonia :  the 
purple  colour  produced  indicates  the  presence  of  urates  in  urine 
(see  Exs.  350  and  352). 

360.  Folin's    method    of    demonstrating    the    presence    of 
uric  acid  in  urine.    To  i  to  2  cc.   (20  drops)   of  urine  in  an 
evaporating  dish  add  one  drop  of  a  saturated  solution  of  oxalic  acid 
and  evaporate  to  complete  dryness  on  a  water-bath.     Allow  to  cool, 
add  10  cc.  of  strong  alcohol  and  allow  to  stand  for  five  minutes  to 
extract  the  polyphenols.    Carefully  pour  off  the  alcohol.    To  the 
residue  add  10  cc.  of  water  and  a  drop  or  two  of  saturated  sodium 
carbonate.    Stir  to  secure  complete  solution  of  the  uric  acid  and 
transfer  to  a  beaker.     Add  i  cc.  of  Folin's  uric  acid  reagent  (Ex. 
354)  and  20  cc.  of  saturated  sodium  carbonate  solution.    The  blue 
colour  that  results  indicates  the  presence  of  uric  acid. 

361.  Urine  has  been  treated  with  about  one-fiftieth  its  bulk 
of  strong  hydrochloric  acid,  and  allowed  to  stand  from  twelve  to 
twenty-four  hours.    Note  the  brown  crystals  of  uric  acid  that  have 
formed  on  the  sides  of  the  vessel.    Examine  them  microscopically : 
they  form  very  irregular  crystals,  usually  arranged  in  sheaves. 
Draw  the  crystals. 

NOTE. — The  chief  pigment  that  associates  itself  with  uric  acid  and  urates 
is  known  as  uroerythrin  (see  p.  278). 


298  URINE.  [CH.  XII. 

G.    Purine  bases,  other  than  uric  acid. 

The  most  important  of  these  found  in  normal  urine 
are  hypoxanthine,  xanthine  and  adenine  (see  p.  62), 
derived  from  the  metabolism  of  food  and  tissue  nucleins  : 
heteroxanthine  (/-methyl-xanthine)  and  paraxanthine 
(i,  7-dimethyl-xanthine)  derived  from  the  breakdown  of 
caffeine  (i,  3,  /-trimethyl-xanthine)  and  theobromine 
(3,  7-dimethyl-xanthine)  of  the  coffee,  tea  and  cocoa 
ingested. 

In  man  the  methylated  xanthines  constitute  the 
greater  part  of  these  purine  bases.  But  it  is  interesting 
to  note  that  the  non-methylated  ones  are  much  increased 
in  fever.  Also  during  severe  muscular  exercise  there  is  an 
increase,  accompanied  by  a  decrease  of  uric  acid.  After 
the  exercise  there  is  an  increase  of  uric  acid,  and  a  decrease 
of  the  other  purines. 

The  simplest  method  of  estimation  is  to  determine 
uric  acid  nitrogen  by  the  method  in  Exs.  394-397,  and 
the  total  purine  nitrogen  by  applying  Kjeldahl's  method 
to  the  total  purines  precipitated  by  ammoniacal  silver 
nitrate  (Ex.  357).  The  difference  is  the  nitrogen  of  the 
purine  bases. 

H.    Creatinine  and  Creatine. 

The  chemical  relationships  of  these  bodies  are  de- 
scribed on  p.  178.  In  normal  human  urine  creatinine  is 
always  present,  but  creatine  only  after  a  meat  diet,  being 
derived  from  that  of  the  food.  Creatine,  however,  is  a 
normal  constituent  of  the  urine  of  children. 

Creatine  seems  to  be  a  product  of  tissue  metabolism, 
and  the  amount  excreted  is  regarded  by  Folin  as  a  measure 
of  endogenous  metabolism.  (See  tables  B  and  C,  p.  270.) 
There  is  an  increase  in  complete  starvation  and  in  fevers, 
due  to  the  increased  tissue  breakdown.  E.  Mellanby 
has  drawn  attention  to  the  fact  that  the  liver  is  probably 
the  seat  of  formation  of  creatinine.  Thus  in  most  diseases 
of  the  liver  there  is  a  decreased  excretion,  an  important 


CH.  XII.]  CREATININE.  299 

exception  being  hepatic  carcinoma,  in  which  condition  the 
urinary-creatinine  is  increased  and  is  accompanied  by 
creatine.  Creatine  is  excreted  when  the  muscles  of  the  body 
are  broken  down.  This  explains  the  presence  of  creatine 
in  urine  during  starvation  and  in  fevers. 

When  creatinine  is  given  by  the  mouth  it  is  mainly 
excreted  unchanged,  but  a  small  portion  is  broken  down 
into  unknown  products.  When  creatine  is  administered 
it  also  is  chiefly  excreted  unchanged,  but  a  certain  per- 
centage is  destroyed  in  the  body.  The  amount  excreted 
unchanged  is  considerably  increased  with  diets  rich  in 
proteins. 

Properties.  Creatinine  dissolves  in  1 1  parts  of  water 
and  1 02  parts  of  alcohol  at  16°  C.  It  is  insoluble  in  ether. 
Its  solutions  are  neutral  or  very  slightly  alkaline  in  reaction. 

Creatinine  is  precipitated  by  phosphotungstic  acid,  by 
picric  acid,  and  by  the  salts  of  the  heavy  metals.  It  forms 
a  characteristic  compound  with  zinc  chloride,  which  is  used 
for  the  preparation  of  standard  solutions. 

Alkalies  convert  it  slowly  into  creatine.  On  boiling 
with  barium  hydroxide  it  is  converted  into  urea  and 
sarcosine  (see  p.  178). 

Creatinine  reduces  Fehling's  solution,  but  not  Bene- 
dict's or  Ny lander's  solutions. 

Creatine  is  converted  to  creatinine  by  heating  with 
acids  (see  Ex.  226).  It  can  be  estimated  by  making 
determinations  of  creatinine  before  and  after  heating  the 
urine  with  acid.  If  aceto-acetic  acid  is  present  Graham 
has  found  that  both  results  are  liable  to  considerable  error 
(see  Graham  and  Poulton,  Proc.  Roy.  Soc.,  LXXXVII.,  B., 
p.  205).  For  the  method  of  estimation  see  p.  336. 

362.    Preparation  of  Creatinine  from  urine.* 

(i.)     Preparation  of  creatinine  pier  ate. 

It  is  best  to  work  on  10  litres  of  urine  at  least.  Dissolve  40  grams,  of  picric 
acid  in  100  cc.  of  boiling  alcohol  and  use  1 8  grams,  of  picric  acid  per  litre  of  urine. 
Pour  the  hot  solution  directly  into  the  urine,  stirring  well  during  the  addition. 

*  (Benedict,  Journal  of  Biological  Chemistry,  xviii.,  p.  183.) 


300  URINE.  [CH.  XII. 

Allow  to  stand  over-night  and  syphon  off  the  supernatant  fluid.  Drain  the 
residue  on  a  Buchner  funnel  and  wash  with  cold  saturated  picric  acid  and 
then  drain  dry. 

(ii.)     Decomposition    of  the   pier  ate. 

Treat  the  dry  creatinine  pi  crate  with  concentrated  hydrochloric  acid  in  a 
mortar,  using  Go  cc.  of  acid  for  every  100  grams,  of  the  picrate.  Stir  thoroughly 
with  the  pestle  for  5  minutes.  Filter  by  suction,  using  a  hardened  filter  paper. 
Wash  residue  twice  with  enough  water  to  cover  it,  sucking  dry  each  time. 
Transfer  the  filtrate  at  once  to  a  large  flask  and  neutralise  with  solid  heavy 
magnesium  oxide.  Add  it  in  small  amounts  at  a  time,  cooling  under  the  tap 
after  each  addition.  The  solution  turns  light  yellow  when  all  the  acid  has  been 
neutralised.  Filter  with  suction  and  wash  the  residue  twice  with  water.  At 
once  add  a  few  cc.  of  glacial  acetic  acid  to  the  nitrate  and  pour  it  into  4  volumes 
of  95  per  cent,  alcohol.  Allow  to  stand  at  least  15  minutes  and  filter  under 
suction. 

(iii.)     Preparation  of  creatinine  zinc  chloride  (CtH^NsO)2.ZnCl2. 

Treat  filtrate  with  a  30  per  cent,  solution  of  zinc  chloride,  using  3-5  cc. 
for  each  litre  of  urine  taken.  Allow  to  stand  over  night  in  a  cool  place.  Pour 
off  the  supernatant  fluid  and  then  collect  the  creatinine  zinc  chloride  in  a 
Buchner.  Wash  once  with  water,  then  thoroughly  with  50  per  cent,  alcohol, 
then  95  per  cent,  alcohol  and  dry.  The  product  should  be  a  nearly  white,  light 
crystalline  powder. 

Yield:     1-2  to  1-5  grams,  per  litre  of  urine. 

(iv.)     Recrystattisation  of  creatinine  zinc  chloride. 

10  grams,  are  treated  with  100  cc.  of  distilled  water  and  then  with  60  cc.  of 
N.  sulphuric  acid.  The  solution  is  heated  to  boiling  till  a  clear  solution  is 
obtained.  About  4  grams,  of  pure  decolourising  charcoal  are  added  and  the 
boiling  continued  for  about  i  minute.  The  solution  is  filtered  off  through  a 
small  Buchner,  the  filtrate  being  refiltered  through  the  same  funnel  two  or 
three  times  till  it  is  quite  clear.  The  residue  is  washed  with  a  little  hot  water 
and  the  total  filtrate  transferred  to  a  beaker.  It  is  then  treated  hot  with  3  cc. 
of  a  strong  solution  of  zinc  chloride  and  about  7  grams,  of  potassium  acetate  dis- 
solved in  a  little  hot  water.  After  10  minutes  the  solution  is  treated  with  an 
equal  volume  of  strong  alcohol  and  allowed  to  stand  for  some  hours  in  a  cool 
place.  The  crystals  are  filtered  off  and  stirred  up  with  about  twice  their 
weight  of  cold  water,  filtered,  washed  with  a  little  water,  and  then  with 
alcohol. 

Yield  :     85  to  90  per  cent,  of  the  crude  material. 

(v.)     Conversion  of  the  zinc  chloride  compound  to  creatinine. 

The  powdered,  recrystallised  compound  is  placed  in  a  dry  flask  and 
treated  with  7  cc.  of  concentrated  aqueous  ammonia  for  every  gram,  taken. 
It  is  slightly  warmed  and  gently  agitated  until  a  clear  solution  is  obtained, 
care  being  taken  to  drive  off  as  little  ammonia  as  possible.  The  flask  is 
then  stoppered  and  placed  in  an  ice  box  for  an  hour  or  two.  Pure  creatinine 
crystallises  out. 

Yield  :     60  to  80  per  cent,  of  the  theoretical. 

362.  Jaffe's  test  for  creatinine.  To  5  cc.  of  urine  add  a  few 
drops  of  a  saturated  aqueous  solution  of  picric  acid  and  of  a  10  per 
cent,  solution  of  sodium  hydroxide.  A  red  colouration  is  produced 
owing  to  the  formation  of  picramic  acid. 


CH.  XII.]  AMMONIA   AND    HIPPURIC   ACID.  3OI 

363.  Weyl's  test  for  creatinine.  To  5  cc.  of  urine  add  a  few 
drops  of  a  freshly  prepared  5  per  cent,  solution  of  sodium  nitro- 
prusside.  Add  a  5  per  cent,  solution  of  sodium  hydroxide,  drop  by 
drop.  A  ruby-red  colour  appears.  Boil.  The  solution  turns 
yellow.  Acidify  with  strong  acetic  acid  and  heat.  A  green  tint 
appears  and  a  blue  precipitate  of  Prussian  blue  may  separate  on 
standing. 

I.    Ammonia. 

Ammonia  is  a  constituent  of  normal  urine,  being 
present  to  the  extent  of  about  0*7  grams,  per  diem.  There 
is  an  increased  excretion  following  the  administration  of 
ammonium  salts  of  inorganic  acids,  in  certain  cases  of 
hepatic  disease,  and  as  a  result  of  acid  poisoning.  This 
last  condition  ("acidosis")  can  be  produced  by  the  adminis- 
tration of  inorganic  acids  or  by  the  excessive  formation  of 
acids  in  the  body,  especially  if  this  is  not  accompanied  by 
an  increased  intake  of  alkalies.  Thus  it  is  seen  in  severe 
diabetes,  in  starvation,  and  in  delayed  chloroform  poison- 
ing, the  acids  formed  being  aceto-acetic  and  /3-oxy-butyric 
acids.  In  certain  forms  of  renal  disease  there  is  a  decreased 
excretion  (see  p.  275). 

For  methods  of  estimation  see  Exs.  398  to  400. 


J.     Hippuric  Acid. 

Hipp  uric  acid  is  formed  in  the  kidney  by  the  con- 
densation of  benzoic  acid  with  glycine. 


C6H5.COOH  +   HaN.CHjj.COOH  =  C6H6.CO.NH.CH2COOH  +  H2O 

Benzoic  acid.  Glycine.  Hippuric  acid. 

The  amount  excreted  by  a  normal  individual  on  a 
mixed  diet  is  about  0-7  grams,  per  diem.  It  is  increased  by  a 
vegetable  diet,  owing  to  the  presence  in  most  plant  foods  of 
an  aromatic  complex  that  is  oxidised  to  benzoic  acid  in  the 
body. 

Hippuric  acid  crystallises  in  4-sided  prisms,  somewhat 
resembling  triple  phosphate.  It  melts  at  187-5°  C.  :  above 


3°2  URINE.  [CH.  XII. 

this  temperature  the  melt  becomes  red  and  is  decomposed 
into  benzoic  acid,  benzonitrile  and  prussic  acid.  It  is 
soluble  in  hot  water,  alcohol  and  ethyl  acetate  :  insoluble 
in  benzene  and  petroleum  ether  :  only  slightly  soluble  in 
cold  water,  alcohol,  ether  and  chloroform.  It  forms  an 
insoluble  ferric  salt.  By  hot  acids  or  alkalies  it  is  hydro- 
lysed  to  benzoic  acid  and  glycine.  When  evaporated  with 
strong  nitric  acid,  nitrobenzene  is  formed. 

364.  Isolation  from  urine  by  Roaf's  method.      500  cc.  of 

the  urine  of  a  horse  or  cow  are  treated  with  125  grams,  of  ammonium 
sulphate  and  7-5  cc.  of  concentrated  sulphuric  acid.  On  standing 
for  24  hours  the  hippuric  acid  crystallises  out.  Filter  off  the 
crystals,  and  wash  with  a  little  cold  water.  Dissolve  in  a  small 
amount  of  hot  water,  boil  with  a  little  adsorbent  charcoal,  filter, 
concentrate  if  necessary,  and  allow  to  stand  for  24  hours. 

365.  To  a  little  hippuric  acid  in  a  small  evaporating  dish  add 
I  to  2  cc.  of  concentrated  nitric  acid  and  evaporate  to  dryness  in 
a  water-bath  in  the  fume  chamber.     Transfer  the  residue  to  a  dry 
test-tube,  apply  heat,  and  note  the  odour  of  nitrobenzene  (artificial 
oil  of  bitter  almonds). 

366.  Neutralise  a  solution  of  hippuric  acid  with  dilute  caustic 
soda.     Add   a   few  drops   of  ferric   chloride.     A   cream-coloured 
precipitate  of  the  ferric  salt  of  hippuric  acid  is  formed. 


K.     Certain  Constituents  of  Abnormal  Urine. 

I.     Albumin  and  Globulin. 

"Albuminuria"  is  the  name  given  to  the  condition  in 
which  a  heat-coagulable  protein  is  found  in  the  urine,  no 
matter  whether  the  protein  present  is  albumin  or  globulin. 
As  a  rule  both  proteins  are  present,  but  albumin  is  gener- 
ally greatly  in  excess  of  the  globulin. 

Albuminuria  can  be  renal  ("true")  or  accidental 
("false").  Renal  albuminuria  can  be  brought  about  by  an 
alteration  in  the  blood  pressure  in  the  kidney,  by  a  change 
in  the  composition  of  the  blood,  or  by  an  alteration  in  the 


CH.  XII.]  ALBUMIN.  303 

structure  of  the  kidney.  In  accidental  albuminuria,  the 
protein  is  not  passed  by  the  kidney,  but  gains  access  to  it 
lower  down  in  the  urinary  tract.  It  is  generally  accom- 
panied by  haemoglobinuria. 

For  routine  work  the  author  uses  the  boiling  test.  In 
cases  of  doubt  the  sulphosalicylic  test  is  simple  and  reliable. 

For  the  method  of  estimating  the  albumin  see  Exs. 
420,421. 

367.  Boiling  test.    Filter  the  urine  till  it  is  clear.     If  it  will 
not  filter  clear,  as  when  infected  with  bacteria,  shake  with  kieselguhr 
and  filter  again.     If  the  urine  be  alkaline  to  litmus,  make  it  faintly 
acid  by  the  cautious  addition  of  i  per  cent,  acetic  acid.     Fill  a 
narrow  test-tube  three  parts  full  with  the  clear  urine,  incline  it  at  an 
angle  and  boil  the  upper  layer  by  means  of  a  very  small  flame.     A 
turbidity  indicates  either  albumin  or  earthy  phosphates  (see  note 
2  to  Ex.  28).     Add  one  or  two  drops  of  strong  acetic  acid,  boiling 
after  the  addition  of  each  drop.     Any  remaining  turbidity  indicates 
the  presence  of  albumin. 

368.  Heller's  test.      Place  about  3  cc.  of  pure  nitric  acid  in  a 
narrow  test-tube.     Float  about  3  cc.  of  filtered  urine  on  the  surface 
of  this,  using  a  pipette  to  avoid  mixing.     A  white  ring  at  the  junc- 
tion of  the  fluids  indicates  the  presence  of  albumin. 

NOTES. — i .  The  white  ring  is  due  to  the  formation  of  metaprotein  by  the 
action  of  the  acid  on  the  albumin,  and  the  insolubility  of  the  metaprotein  in 
the  strong  nitric  acid  (see  Exs.  21  and  40). 

2.  A  coloured  ring  is  usually  produced  owing  to  the  oxidation  of  certain 
urinary  chromogens. 

3.  In  very  concentrated  urine,  a  white  ring  of  urea  nitrate  may  form. 
It  usually  has  very  sharply  denned  borders. 

4.  If  the  urine  is  very  rich  in  urates,  a  precipitate  of  uric  acid  may  form 
at  the  junction  of  the  fluids,  or,  more  commonly,  somewhat  above  the  nitric 
acid.     Urea  and  uric  acid  are  distinguished  from  albumin  by  the  previous 
dilution  of  the  urine  with  two  or  three  volumes  of  water. 

5.  The  presence  of  resinous  substances  in  the  urine  of  patients  who  have 
been  treated  with  balsams  leads  to  the  development  of  a  white  ring  or  cloud 
that  disappears  on  treatment  with  alcohol. 

6.  Urine  rich  in  albumose  may  give  a  white  cloud  that  disappears  on 
warming. 

7.  Urine  that  has  been  preserved  by  the  addition  of  thymol  gives  a  ring 
of  nitroso thymol  or  nitro thymol.     The  thymol  can  be  removed  by  gentle 
agitation  with^petroleum  ether. 


304  URINE.  [CH.  XII. 

369.  Roberts9  test.     Repeat   the    previous    exercise,    using 
Roberts'  reagent  in  place  of  the  nitric  acid.     A  white  ring  at  the 
junction  of  the  fluids  indicates  albumin. 

NOTES. — i.     Roberts'  reagent  is  prepared  by  adding  I  volume  of  pure 
nitric  acid  to  5  volumes  of  a  saturated  solution  of  magnesium  sulphate. 

2.     Coloured  rings  are  not  formed,  and  so  confusion  is  avoided. 

370.  Spiegler's  test.      Render  the  urine  faintly  acid  with 
acetic  acid  and  repeat  the  above  test,  using  Spiegler's  reagent  in 
place  of  Roberts*.     A  white  ring  indicates  the  presence  of  albumin. 


NOTES. — i.     Spiegler's  reagent  consists  of 
Mercuric  chloride 
Tartaric  acid 
Glycerine 
Sodium  chloride 
Distilled  water      .. 


40  grams. 

20  grams. 

100  grams. 

50  grams. 

1000  cc. 


2.  The  reaction  is  also  given  by  albumoses  and  peptones. 

3.  The  test  serves  to  show  i  part  of  albumin  in  250,000.     It  is  almost  too 
delicate  for  ordinary  clinical  work,  as  a  large  number  of  apparently  normal 
urines  give  a  positive  reaction. 

371.  Sulphosalicylic  test.     To  a  few  cc.  of  the  clear,  filtered 
urine  add  a  large  "  knife  point  "  or  a  few  drops  of  a  20  per  cent, 
solution  of  sulphosalicylic  acid  (see  Ex.  18).     A  cloud  or  precipitate 
indicates  the  presence  of  albumin. 

2.     Albumoses. 

Albumoses  are  found  in  the  urine  in  certain  cases  of 
degeneration  of  the  intestinal  epithelium  ("alimentary 
albumosuria").  Also  in  a  variety  of  other  conditions  such 
as  in  the  absorption  of  pneumonic  exudates,  in  some  cases 
of  an  increased  breakdown  of  the  tissues  in  certain  fevers, 
in  the  puerperium  and  in  urine  containing  semen. 

The  albumose  present  seems  to  be  a  secondary  album  ose. 

372.  Remove  any  albumin  that  may  be  present  by  heat 
coagulation.    To  the  filtrate  apply  Spiegler's  test  (Ex.  280).    A 
white  ring  indicates  the  presence  of  albumose. 

j.     Bence- Jones*  Protein. 

In  certain  cases  of  disease  of  the  bone  marrow  (multiple 
myeloma),  and  possibly  in  osteomalacia,  a  protein  with 


CH.  XII.]  BENCE-JONES'    PROTEIN.  305 

peculiar  properties  is  found  in  the  urine.  It  is  named 
after  Bence-Jones,  who  first  described  the  condition.  It 
has  the  property  of  coagulating  at  temperatures  under 
55°  C.,  of  redissolving  to  a  clear  solution  on  boiling  and 
of  reappearing  on  cooling.  It  is  precipitated  by  half- 
saturation  with  ammonium  sulphate.  It  is  not  precipi- 
tated on  dialysis. 

Hopkins  has  shewn  that  the  solution  of  the  heat 
coagulum  on  boiling  depends  on  the  presence  of  neutral 
salts,  those  with  divalent  cations  (as  CaCl2)  being  most 
potent  in  neutral  or  faintly  acid  solutions,  and  those  with 
divalent  anions  (as  K2SO4)  in  faintly  alkaline  solutions. 

Hopkins  has  also  shewn  that  the  protein  excreted  is 
formed  in  the  body,  either  in  the  marrow  or  as  a  result  of 
the  influence  of  the  growth  on  general  metabolism.  The 
amount  in  the  urine  is  independent  of  the  nature  or  amount 
of  the  proteins  of  the  food.  The  nitrogen  of  the  protein 
excreted  may  be  as  high  as  one-third  of  the  total  urinary 
nitrogen. 

373.  If  necessary  make  the  suspected  urine  faintly  acid  with 
acetic  acid.  Heat  carefully  by  immersing  in  a  beaker  of  warm 
water.  The  urine  becomes  turbid  at  40°  to  45°  C.,  and  shows  a 
flocculent  precipitate  at  60°  C.  On  raising  the  temperature  to  ioo°C. 
the  precipitate  partially  or  completely  disappears.  On  cooling  it 
reappears. 

4.     Blood  Pigments. 

Blood  pigments  may  occur  in  pathological  urine  in 
intact  corpuscles  ("haematuria")  or  free  in  solution 
(' '  haemoglobinuria  "). 

Haematuria  can  be  recognised  by  determining  the 
presence  of  red  corpuscles  by  a  microscopic  examination 
of  the  sediment  obtained  by  centrifugalising  the  urine. 
It  occurs  with  gross  lesions  of  the  kidney  or  ^ay  part 
of  the  urinary  tract,  so  that  blood  passes  directly  into  the 
urine.  If  the  blood  comes  from  the  kidney  it  is  well 
mixed  with  the  urine.  If  the  blood  comes  from  the  bladder 

w 


306  URINE.  [CH.  XII. 

or  genital  organs  it  often  forms  a  clot.  In  haematuria  the 
urine  often  has  a  characteristic  smoky  appearance,  and  it 
is  always  associated  with  albuminuria.  Haemoglobinuria 
is  a  result  of  haemolysis.  It  therefore  follows  a  variety 
of  infectious  diseases,  transfusion  of  blood,  the  absorption 
of  haemolytic  substances,  such  as  many  aromatic  com- 
pounds, severe  burns  and  scalds.  Methaemoglobin  is 
nearly  always  present. 

The  simplest  method  of  detecting  blood  is  by  means 
of  the  benzidine  test,  provided  that  the  necessary  reagents 
are  to  hand. 

374.  Heller's  test.     Boil  10  cc.  of  urine  with  a  little  40 
per  cent,  sodium  hydroxide,  and  allow  the  tube  to  stand  for  a  while. 
A  red  deposit  indicates  the  presence  of  blood-pigment  in  the  urine. 
Pour  off  the  supernatant  fluid  and  acidify  with  acetic  acid.     The 
precipitate  dissolves  only  partially,  leaving  a  red  residue. 

NOTES. — i.  The  alkali  converts  the  pigment  into  haematin,  which  is 
precipitated  with  the  earthy  phosphates. 

2.  Certain  substances,  such  as  cascara  sagrada,  rhubarb,  senna  and 
santonin  cause  the  urine  to  give  a  similar  red  precipitate  when  boiled  with 
alkali.  But  in  these  cases  the  precipitate  dissolves  completely  in  acetic  acid. 

375.  Schumm's  spectroscopic  test.    Treat  50  cc.  of  the  urine 
with  5  cc.  of  glacial  acetic  acid  and  50  cc.  of  ether.     Shake  thoroughly 
in  a  separating  funnel.     Allow  to  stand  and  add  a  drop  or  two  of 
alcohol  to  obtain  a  separation  of  the  layers.     Run  off  the  urinary 
layer.    To  the  ether  add  5  cc.  of  water,  shake  and  run  off  the  water. 
To  the  washed  ether  add  ammonia  and  shake  for  half  a  minute, 
cooling  under  the  tap.     The  reaction  must  be  markedly  alkaline 
after  shaking.     Run  off  the  lower  coloured  layer  into  a  tube,  add 
5  to  10  drops  of  ammonium  sulphide  solution  and  examine  spectro- 
scopically  for  the  bands  of  haemochromogen.     (Ex.  305.) 

376.  Benzidine  test.     To  a  large  "  knife  point  "  of  benzidine 
in  a  perfectly  clean,  dry  test-tube  add  about  3  cc.  of  glacial  acetic 
acid  and  agitate  for  about  a  minute.     Add  an  equal  volume  of 
"  10  volumes  "  hydrogen  peroxide.     Mix  and  pour  one-half  into 
another  clean,  dry  test-tube.    To  one  of  the  tubes  add  I  cc.  of  the 


CH.  XII.]  BILE   PIGMENTS.  307 

suspected  urine.  The  fluid  rapidly  acquires  a  deep  blue  tint  if 
blood  pigment  is  present.  Should  the  untreated  fluid  also  develop 
a  blue  tint,  the  test  should  be  repeated,  the  control  tube  being  treated 
with  i  cc.  of  a  normal  urine.  By  following  this  procedure  the  test 
is  a  very  conclusive  one.  The  reaction  can  be  applied  to  an  acid 
ether  extract  prepared  by  the  method  given  in  the  above  exercise. 


5.     Bile. 

The  constituents  of  the  bile  are  found  in  urine  when 
the  bile  duct  is  obstructed  by  a  calculus  or  by  catarrh. 
The  bile  is  absorbed  into  the  lymphatics,  passes  into  the 
circulation  and  reaches  all  parts  of  the  body,  the  pigments 
causing  a  staining  of  the  various  tissues.  The  condition 
is  known  as  jaundice. 

The  absence  of  bile  salts  from  the  urine  does  not 
exclude  the  possibility  of  the  presence  of  bile  pigments. 
With  continued  obstruction  of  the  bile  passages  the 
formation  of  bile  salts  seems  to  decrease.  Urine  contain- 
ing bile  often  has  a  characteristic  appearance. 

377.  Cole's  test  for  bile  pigments.  Treat  10  to  15  cc. 
of  the  urine  with  2  drops  of  saturated  magnesium  sulphate  and 
proceed  as  directed  in  Ex.  318.  If  a  hand  centrifuge  is  available,  the 
test  is  more  sensitive  if  an  excess  of  barium  chloride  is  added  to  the 
unheated  urine  and  the  precipitate  driven  drown  by  spinning  in  the 
machine.  The  supernatant  fluid  is  poured  off  as  cleanly  as  possible, 
the  precipitate  stirred  with  the  alcohol  and  sulphuric  acid,  trans- 
ferred to  a  test-tube  and  boiled  with  the  potassium  chlorate. 

In  a  certain  number  of  cases  the  result  is  obscured  by  the 
presence  of  certain  other  pigments.  In  such  cases  to  render  the 
test  more  delicate,  pour  off  the  alcoholic  solution  from  the  barium 
sulphate  into  a  dry  tube.  Add  about  one-third  its  volume  of 
chloroform  and  mix.  To  the  solution  add  about  an  equal  volume 
of  water,  place  the  thumb  on  the  tube,  invert  once  or  twice  and 
allow  the  chloroform  to  separate.  It  contains  the  bluish  pigment 
in  solution. 


308  URINE.  [CH.  XII. 

378.  Hay's   test   for    bile    salts.     Sprinkle  the   surface   of 
some  urine  in  a  test-tube  with  flowers  of  sulphur.     The  particles 
fall  to  the  bottom  of  the  tube  if  bile  salts  are  present.    (See  Ex.  316.) 

379.  Oliver's  test  for  bile  salts.     Acidify  the  urine  with 
acetic  acid  and  filter  if  necessary.     To  it  add  a  clear  i  per  cent, 
solution  of  Witte's  peptone,  also  acidified  with  acetic  acid.     A  white 
precipitate  indicates  bile  salts.     (Ex.  317.) 

6.     Glucose. 

Glucose  is  not,  strictly  speaking,  an  abnormal  con- 
stituent of  urine.  The  author  was  finally  convinced  of 
this  some  years  ago  when  working  at  a  method  for  the 
detection  of  small  amounts  of  glucose  in  urine*  (see  Ex.  381). 
Folinf  confirmed  this,  using  practically  the  same  method. 
Recently  Benedict  and  Osterbergf  have  introduced  a  new 
method  for  the  estimation  of  glucose  in  normal  urine  (see 
Ex.  407),  and  although  it  has  only  been  applied  to  a  few 
individuals,  the  results  obtained  are  of  very  great  im- 
portance, and  will  probably  serve  as  the  starting  point  for 
a  new  attack  on  the  problems  of  diabetes.  According  to 
these  observers  about  i  gram,  of  non-nitrogenous  reducing 
substance  is  excreted  per  diem,  of  which  about  55  per  cent, 
is  not  fermentable  by  yeast,  and  has  not  yet  been  identified. 
The  effect  of  diet  is  interesting.  The  excretion  is  increased 
by  carbohydrate  intake,  especially  at  breakfast.  A 
similar  intake  at  mid-day,  during  normal  muscular  activity, 
has  a  much  smaller  effect.  For  this  reason  a  normal 
individual  may  pass  a  urine  shortly  after  breakfast  which 
might  cause  him  to  be  rejected  as  a  diabetic  when  examined 
for  life  insurance.  Such  cases  would  probably  Be  passed  as 
normal  if  a  sample  of  the  mixed  24  hours'  specimen  were 
examined.  The  effect  of  taking  glucose  varies  with  the 
dose  and  also  with  the  time  of  administration.  Apparently 

*  Cole,  Lancet,  1913,  ii.,  p.  859. 

f  Folin,  Journ.  Biol.  Chem.,  xxii.,  p.  327. 

J  Benedict  and  Osterberg,  Journ.  Biol.  Chem.,  xxxiv.,  p.  195. 


CH.  XII.]  GLUCOSE.  309 

glucose  is  tolerated  better  on  an  empty  stomach  than  when 
taken  with  an  ordinary  meal.  In  general,  it  might  be 
stated  that  a  normal  individual  should  be  able  to  absorb 
50  grams,  of  glucose  on  an  empty  stomach  without  showing 
any  increase  in  the  amount  of  sugar  excreted  in  a  given 
time.  An  important  result  of  these  researches  is  that  the 
concentration  of  sugar  in  the  urine  is  of  much  less  signifi- 
cance than  the  amount  passed  per  hour.  Since  urine 
always  contains  glucose,  they  suggest  that  the  term 
"glycuresis"  should  replace  "glycosuria,"  to  indicate 
conditions  characterised  by  an  increased  excretion  of 


glucose  in  the  urine. 


There  are  two  types  of  glycuresis,  alimentary  and 
persistent.  Alimentary  glycuresis  is  the  condition  in 
which  the  amount  of  sugar  absorbed  exceeds  the  amount 
that  the  individual  is  capable  of  assimilating.  The  limit 
varies  with  the  individual,  and  is  affected  by  a  variety  of 
pathological  conditions.  Persistent  glycuresis  is  the  condi- 
tion when  large  amounts  of  sugar  are  excreted  for  a  con- 
siderable length  of  time,  and  may  be  quite  independent 
of  the  administration  of  carbohydrate  food.  The  condition 
is  known  as  diabetes  mellitus.  The  urine  is  generally 
much  increased  in  amount,  of  a  high  specific  gravity,  and 
pale  in  colour. 

The  classical  test  for  sugar  in  urine  is  Fehling's  (Ex.  97).  It  is  not  a 
reliable  test.  Not  only  is  Fehling's  solution  reduced  by  certain  constituents  of 
normal  urine,  such  as  urates  and  creatinine  ;  but  also  certain  of  these  bodies, 
notably  creatinine,  form  soluble  compounds  with  cuprous  oxide,  and  thus 
markedly  interfere  with  the  delicacy  of  the  test.  Also  the  urea  of  the  urine  is 
decomposed  to  ammonia,  which  dissolves  cuprous  oxide  (see  p.  106,  note  5). 
Further,  glucose  is  destroyed  by  boiling  caustic  soda,  so  that  the  presence  of  a 
small  amount  of  sugar  may  escape  detection. 

Benedict's  test  (Ex.  100)  is  a  great  improvement.  Owing  to  the  substitu- 
tion of  sodium  carbonate  for  sodium  hydroxide  the  solution  is  not  reduced  by 
urates  or  creatinine.  It  does  not  give  a  positive  reaction  with  the  concentra^ 
tion  of  glucose  normally  present  in  urine,  but  is  very  sensitive  for  small  in- 
creases beyond  this.  The  author  considers  it  the  most  reliable  for  general  use ; 
but  owing  to  the  fact  that  it  is  much  more  delicate  than  Fehling's,  the  result 
of  a  faintly  positive  test  is  not  necessarily  an  alarming  indication  of  abnor- 
mality. 

Cole's  test  (Ex.  381)  is  more  sensitive  than  Benedict's,  and  the  manipula- 
tion has  been  so  arranged  as  to  ensure  that  it  does  not  give  a  positive  result 
with  normal  urines.  It  is  of  considerable  value  in  detecting  small  variations 


310  URINE.  [CH.  XII. 

from  the  normal,  but  such  cases  should  -be  examined  by  the  application  of 
Benedict  and  Osterberg's  quantitative  method.     ^£x..  407  J 


The  osazone  test  serves  to  confirm  the  presence  of  glucose  in  doubtful 
cases,  and  especially  to  distinguish  between  glucose  on  the  one  hand  and 
lactose  and  pentoses  on  the  other. 

The  fermentation  test  is  helpful  in  connexion  with  the  recognition  of 
lactose  and  glycuronic  acid. 

380.  Benedict's  test.     To  5  cc.  of  Benedict's  reagent  (see 
p.  107)  in  a  test-tube  add  eight  drops  of  the  urine.     Boil  vigorously 
for  two  minutes  and  allow  to  cool  spontaneously.     If  glucose  is 
present  the  entire  body  of  the  solution  will  be  filled  with  a  precipitate 
which  may  be  red,  yellow  or  green  in  colour,  depending  on  the 
amount  of  sugar. 

NOTE.  —  It  is  essential  to  add  a  small  volume  of  urine.  If  too  much  be 
added  the  results  are  apt  to  be  ambiguous.  Even  with  the  eight  drops 
recommended,  a  slight  precipitate  of  earthy  phosphates  may  appear  and 
simulate  a  feeble  reduction. 

381.  Cole's  test  for  small  amounts  of  glucose  in  urine.    In 

a  dry  boiling  tube  or  large  test-tube  place  about  I  gram,  of  adsorbent 
charcoal.  Add  10  cc.  of  the  urine,  shake,  heat  to  boiling  and  then 
cool  under  the  tap.  Shake  at  intervals  for  5  minutes.  Filter 
through  a  small  paper  into  a  dry  test-tube.  To  the  filtrate  add  4 
drops  of  pure  glycerol  and  0-5  gram,  of  anhydrous  sodium  carbonate. 
Shake  and  heat  to  boiling.  Maintain  the  boiling  for  exactly  50  sees. 
Immediately  add  4  drops  of  a  5  per  cent,  solution  of  crystalline 
copper  sulphate,  shake  to  mix  and  allow  the  tube  to  stand  without 
further  heating  for  one  minute.  With  normal  urine  the  fluid 
remains  blue.  If  glucose  is  present  to  the  extent  of  0*03  per  cent. 
above  the  normal  amount  in  urine  the  blue  colour  is  discharged  and 
a  yeUowish  precipitate  of  cuprous  hydroxide  forms. 

NOTES.  —  i.  Treatment  with  adsorbent  charcoal  removes  practically  the 
whole  of  the  urates,  creatinine  and  pigments  that  interfere  with  Fehling's  test. 
It  also  adsorbs  so  much  of  the  normal  amount  of  glucose  present  that  the 
nitrate  from  normal  urine  fails  to  give  a  reduction. 

2.  0-5  gram,  of  anhydrous  sodium  carbonate  is  carried  by  about  f  the 
length  of  a  large  blade  well  piled  up  once. 

3.  Should  the  specific  gravity  of  the  urine  exceed  1025  it  is  advisable  to 
use  5  cc.  of  the  urine  +  5  cc.  of  water. 

4.  The  test  is  not  given  by  chloroform  nor  by  glycuronates  :  it  is  given  by 
pentoses. 


CH.  XII.]  GLUCOSE.  3H 

5.  Should  there  be  any  reason  to  suspect  lactose  the  procedure  should  be 
modified  as  follows  :  treat  20  cc.  of  the  urine  with  i  gram,  of  charcoal  as  de- 
scribed above.  Treat  the  whole  of  the  nitrate  with  another  gram,  of  charcoal 
and  repeat  the  process.  To  5  cc.  of  this  nitrate  add  the  glycerol  and  sodium 
carbonate  and  proceed  as  above  directed.  A  reduction  indicates  the  presence 
of  glucose,  the  whole  of  any  lactose  up  to  even  I  per  cent,  being  removed  by 
this  double  adsorption,  whilst  0-04  per  cent,  of  glucose  in  the  original  urine  still 
shows  in  the  nitrate. 

382.  Fehling's  test.      Boil  3  to  5  cc.  of  Fehling's  solution 
(see  p.  1 06)  to  ascertain  whether  the  Rochelle  salt  has  been  de- 
composed into  reducing  substances.     If  no  reduction  occurs  boil 
the  same  volume  of  urine  in  another  tube.     Reboil  the  Fehling's 
solution  and  mix  the  two.     Allow  the  tube  to  stand  without  further 
heating.     If  any  appreciable  amount  of  glucose  is  present  a  red  or 
yellow  precipitate  will  appear. 

NOTE. — Prolonged  boiling  of  the  urine  with  Fehling's  solution  is  very 
apt  to  lead  to  the  formation  of  a  greenish-yellow  precipitate  owing  to  the 
action  of  the  strong  alkali  on  the  normal  urinary  constituents. 

383.  Phenylhydrazine  test.     Treat  10  cc.  of  urine  with  i  cc. 
of  strong  acetic  acid.     Add  enough  phenylhydrazine  hydrochloride 
to  cover  a  sixpenny  piece  and  twice  this  bulk  of  solid  sodium  acetate. 
Dissolve  by  the  aid  of  heat  and  filter.     Place  the  filtrate  in  a  tube 
and  immerse  this  in  a  boiling  water-bath  for  30  to  60  minutes. 
Turn  out  the  flame  and  allow  the  tube  to  cool  without  removing  it 
from   the   bath.     Examine   the   deposit   microscopically   for   the 
characteristic  crystals  of  glucosazone  (see  p.  no). 

NOTE. — With  small  amounts  of  glucose  the  crystals  are  apt  to  separate 
in  small  spherical  clusters. 

384.  Fermentation  test.      Fill  a  test-tube  with  urine  and 
then  transfer  the  fluid  to  a  mortar.     Add  a  piece  of  washed  yeast 
about  the  size  of  a  bean  and  pound  it  up  with  the  urine.     Transfer 
the  mixture  to  the  test-tube  and  invert,  placing  the  open  end  under 
mercury  or  urine  contained  in  a  small  dish.     Clamp  the  tube  in 
position,  and  allow  it  to  stand  for  at  least  eighteen  hours  in  a  warm 
place.     If  glucose  is  present  in  the  urine  there  is  an  accumulation 
of  gas  (CO2)  at  the  top  of  the  tube. 

NOTES. — i.  Lactose,  pentoses  and  glycuronic  acids  are  not  fermented  by 
pure  yeast. 

2.  A  special  apparatus  called  Einhorn's  saccharometer  has  been  devised 
to  enable  the  test  to  be  applied  conveniently.  Also  the  volume  of  CO2  formed, 
and  the  percentage  of  glucose  present  can  be  roughly  determined  by  means 
of  it. 


312  URINE.  [CH.  XII. 

7.     Fructose  (laevulose). 

Fructose  occasionally  occurs  in  the  urine,  sometimes 
being  accompanied  by  glucose.  The  significance  of  fructo- 
suria  is  not  yet  clear. 

385.  Seliwanoff's  test  (Borchardt's  modification).    To  a  few 
cc.  of  urine  in  a  test-tube  add  an  equal  volume  of  25  per  cent, 
hydrochloric  acid  and  a  speck  or  two  of  resorcin.     Heat  to  boiling, 
cool  under  the  tap,  and  transfer  to  an  evaporating  dish.     Make  the 
reaction  alkaline  by  means  of  solid  sodium  hydroxide  and  return  it 
to  a  test-tube.     Add  3  cc.  of  acetic  ether  (ethyl  acetate)  and  shake. 
A  yellow  colouration  in  the  acetic  ether  indicates  the  presence  of 
fructose. 

8.     Pentoses. 

Pentoses,  that  is  carbohydrates  with  5  carbon  atoms, 
appear  in  the  urine  in  three  conditions,  alimentary, 
persistent  or  true  pentosuria,  and  admixed  with  glucose  in 
cases  of  glycuresis. 

Alimentary  pentosuria  is  sometimes  seen  after  the 
mgestion  of  considerable  quantities  of  certain  fruits,  as 
prunes,  cherries,  grapes  and  plums.  The  sugar  found 
varies,  but  is  usually  d-arabinose.  In  true  pentosuria  it 
is  ^/-arabinose.  Its  origin  and  significance  have  not  yet 
been  clearly  established. 

Pentoses  are  indicated  when  the  urine  gives  a  positive 
Benedict's  or  Cole's  test,  but  a  negative  fermentation  test. 
The  results  of  the  phenyl-hydrazine  test  are  variable,  the 
osazones  being  somewhat  soluble.  The  two  colour  re- 
actions described  are  also  given  by  glycuronic  acid,  which 
can,  however,  be  demonstrated  by  Ex.  392. 

386.  Tollen's  test.      To  5  cc.  of  urine  add  an  equal  volume 
of  strong  hydrochloric  acid  and  a  little  phloroglucin  (a  piece  about 
the  size  of  a  pea)  and  heat  the  mixture  on  a  boiling  water  bath. 
A  cherry-red  colour  develops  and  the  solution  shows  an  absorption 
band  between  D  and  E.     On  cooling  a  dark  precipitate  separates 


CH.  XII.]  TOTAL    NITROGEN.  313 

out.     On  dissolving  this  in  strong  alcohol,  the  alcoholic  solution 
shows  the  colour  and  absorption  band  of  the  original  mixture. 

387.  Bial's  orcin  test.    To  2  -  3  cc.  of  urine  add  4  -  5  cc.  of 
Bial's  reagent  and  heat  till  boiling  commences.     A  green  colour  or 
the   formation   of   a   green   precipitate   indicates   pentoses.     The 
solution  shows  two  absorption  bands,  one  in  the  red  between  B  and 
C  and  the  other  near  the  D  line. 

NOTE. — Bial's  reagent  consists  of  i  to  1-5  gram,  orcine,  500  cc.  of  concen- 
trated hydrochloric  acid,  and  30  drops  of  a  i  per  cent,  solution  of  ferric 
chloride. 

9.     Lactose. 

Lactose  is  found  in  the  urine  of  women  during  pregnancy, 
during  the  nursing  period,  and  soon  after  weaning.  The 
amount  in  the  urine  varies,  but  rarely  exceeds  i  per  cent. 
The  excretion  usually  reaches  its  maximum  2  to  4  days 
after  parturition. 

The  only  satisfactory  method  of  demonstrating  the 
presence  of  lactose  in  urine  is  by  the  author's  test.  The 
mucic  acid  test  is  inconvenient  and  not  very  reliable. 

388.  Cole's  test  for  lactose.      To  i  gram,  of  good  charcoal 
add  25  cc.  of  the  suspected  urine,  mix  by  shaking,  boil  for  a  few 
seconds,  cool  thoroughly,  and  shake  at  intervals  for  10  minutes. 
Filter  through  a  small  paper  or  use  a  filter  pump.     When  the  char- 
coal has  completely  drained  transfer  it  to  a  porcelain  dish  containing 
10  cc.  of  water  and  i  cc.  of  glacial  acetic  acid.     This  is  best  done  by 
opening  the  paper,  holding  it  by  the  clean  half  and  moving  it  about 
in  the  liquid.     The  greater  part  of  the  charcoal  is  thus  removed  from 
the  paper.     Stir  the  charcoal  with  a  glass  rod  and  transfer  the 
mixture  to  a  boiling  tube.     Heat  to  boiling  for  about  10  seconds  and 
filter  the  hot  solution  through  a  small  paper  into  a  test-tube  contain- 
ing as  much  solid  phenyl-hydrazine-hydrochloride  as  will  lie  on  a 
shilling,   and  twice  this  amount  of  solid  sodium  acetate.     Mix 
thoroughly  and  filter  from  any  insoluble  oily  residue.     Place  the 
tube  in  a  boiling  water  bath  and  leave  it  there  for  45  minutes. 
Remove  the  tube  and  allow  it  to  stand  at  room  temperature  for  at 
least  one  hour.     It  is  advisable  to  allow  it  to  stand  longer  if  possible. 


314  URINE.  [CH.  XII. 

Pipette  off  a  little  of  the  deposit,  if  any,  and  examine  it  on  a  slide 
under  the  high  power  of  a  microscope. 

Lactosazone  crystallises  in  characteristic  clumps  with  project- 
ing spines  ("hedge-hog"  crystals).  It  can  be  recrystallised  by 
filtering  through  a  small  paper,  washing  with  a  small  amount  of 
distilled  water,  and  then  passing  about  4  cc.  of  boiling  water  through 
the  paper  into  a  clean  tube.  The  filtrate  is  boiled  and  passed 
through  the  paper  two  or  three  times,  boiling  between  every  filtra- 
tion. On  allowing  the  solution  to  stand,  typical  crystals  of  the 
osazone  separate  out. 

389.  Mucic  acid  test.  TOO  cc.  of  the  urine  and  20  cc.  of  pure 
concentrated  nitric  acid  are  evaporated  in  a  wide  and  rather  shallow 
beaker  on  a  boiling  water  bath  in  a  fume  chamber.  The  evaporation 
is  continued  until  the  fluid  becomes  clear,  and  brown  fumes  are  no 
longer  evolved.  The  total  volume  is  then  about  20  cc.  Remove 
the  beaker  from  the  bath  and  transfer  the  contents  to  a  smaller 
beaker,  washing  out  with  a  small  amount  of  distilled  water.  Allow 
to  stand  over-night  in  a  cool  place.  The  formation  of  a  white 
crystalline  mass  of  mucic  acid  indicates  the  presence  of  lactose  in  the 
urine.  Dilute  the  fluid,  collect  the  crystals  on  a  small  filter  and 
wash  with  cold  water.  Microscopically  the  crystals  are  seen  to  be 
very  pointed  prisms  with  oblique  angles.  The  melting  point  is 
213°  —  215°  C.  It  can  be  weighed  and  titrated  with  standard 
alkalies,  its  equivalent  weight  being  105. 

NOTE.— Mucic  acid  is  COOH.(CHOH)4COOH. 

10.     The  Acetone  bodies. 

The  acetone  bodies  found  in  urine  in  certain  forms  of 
the  condition  known  as  "  acidosis  "  are 
Acetone.     CH3.CO.CH3. 
Aceto-acetic  acid.     CH3.CO.CH2.COOH. 
/3-oxy-butyric   acid   CH3.CH(OH).CH2.COOH. 

The  acetone  must  be  regarded  as  a  decomposition 
product  of  aceto-acetic  acid,  which  loses  CO2  on  being 
heated.  The  excretion  of  the  acetone  bodies  depends 
on  the  inability  of  the  tissues  to  oxidise  completely  the 


CH.  XII.]  ACETONE  BODIES.  315 

fatty  acids  generally  derived  from  the  fats,  but  sometioaes 
from  certain  of  the  amino-acids  formed  in  the  metabolism 
of  proteins.  The  condition  that  usually  gives  rise  to 
acetonuria  is  the  inability  of  the  tissues  to  obtain  or  to 
utilise  an  adequate  amount  of  glucose.  Thus  these  acetone 
bodies  are  excreted  in  starvation,  on  a  diet  of  fats  with  a 
limited  amount  of  protein,  in  certain  fevers,  severe  anaemias 
and  after  phosphorus  poisoning,  and  finally  in  diabetes 
mellitus,  in  which  condition  the  tissues  are  unable  to 
utilise  the  glucose  provided. 

It  is  a  remarkable  fact  that  the  urine  passed  just  after 
an  operation  with  a  volatile  anaesthetic  nearly  always 
contains  a  considerable  amount  of  the  acetone  bodies. 
The  author  has  confirmed  Piper's  statement  that  this 
post-operative  acidosis  is  much  decreased  by  the  previous 
administration  of  dried  pancreatic  tissue  ("pancreatin"). 
It  is  also  noteworthy  that  the  acidosis  is  partially  depen- 
dent on  the  previous  dietetic  treatment  of  the  patient,  a 
fair  dose  of  a  carbohydrate  such  as  lactose  having  a  very 
beneficial  effect.  In  some  cases,  especially  after  the 
use  of  chloroform,  the  acidosis  may  recur.  This  condition 
of  "  delayed  chloroform  poisoning  "  is  very  apt  to  lead  to 
fatal  results. 

In  children  recurrent  vomiting  may  be  associated  with 
acidosis.  In  many  of  these  cases  the  condition  of  the 
patient  is  similar  to  that  seen  in  peritonitis  or  appendicitis. 
Examination  often  reveals  a  deep  pain  on  pressure  over 
the  pancreas.  Operative  interference  in  such  cases  without 
treatment  of  the  acidosis  is  an  extremely  risky  under- 
taking. The  recognition  of  the  condition  of  acetonuria 
is  therefore  of  great  importance. 

The  simplest  and  most  reliable  test  for  acetone  and 
aceto-acetic  acid  is  Rothera's.  So  far  a  simple  test  for 
/3-oxy-butyric  acid  has  not  been  introduced.  But  as  this 
is  only  found  together  with  aceto-acetic  acid,  its  presence 
is  only  of  qualitative  significance. 

The  methods  of  estimation  of  the  acetone  bodies  are 
described  on  pages  347  to  352. 


316  URINE.  [CH.  XII. 

389.  Rothera's  test  for  acetone  and  aceto-acetic  acid.    To 

10  cc.  of  the  urine  add  an  excess  of  solid  ammonium  sulphate,  so 
that  the  urine  is  completely  saturated.  Then  add  two  or  three 
drops  of  a  freshly  prepared  5  per  cent,  solution  of  sodium  nitro- 
prusside  and  2  or  3  cc.  of  concentrated  ammonia.  Mix  and  allow  to 
stand  undisturbed  for  at  least  thirty  minutes.  A  characteristic 
permanganate  colouration,  that  may  only  develop  above  the  layer 
of  undissolved  crystals,  indicates  the  presence  of  acetone  or  aceto- 
acetic  acid. 

NOTES. — i.  The  test  is  much  more  sensitive  for  aceto-acetic  acid  than 
for  acetone.  The  reaction  with  acetone  can  just  be  detected  in  a  dilution  of 
i  in  20,000,  whilst  aceto-acetic  acid  shows  it  in  a  i  in  400,000  dilution. 

2.  The  intensity  of  the  colour  and  the  rate  at  which  it  develops  vary 
with  the  concentration.     Kennaway*  suggests  the  following  scale  : — (i)  quick- 
strong  ;   (2)  slow-strong  ;   (3)  quick-weak  ;   (4)  slow- weak.     A  "  quick-strong  " 
reaction  shows  a  deep  permanganate  colour  within  a  few  seconds,  and  indicates 
the  presence  of  at  least  0-25  per  cent,  aceto-acetic  acid.     A  "  slow-weak  " 
reaction  may  show  no  pink  for  some  minutes,  and  the  amount  present  is 
probably  less  than  0-0005  Per  cent. 

3.  Though  aceto-acetic  acid  gives  a  vivid  reaction,  aceto-acetic  ester 
actually  inhibits  the  reaction  when  the  free  acid  or  acetone  are  present. 

4.  For  the  sake  of  experience  it  is  advisable  to  practise  the  tests  on 
urines  containing  varying  amounts  of  acetone  and  aceto-acetic  acid.     The 
latter  is  prepared  from  the  ester  by  the  following  method  of  Hurtley. 

Into  a  beaker  weigh  out  13  grams,  (i-io  molecule)  of  aceto-acetic  ester. 
Add  100  cc.  of  N.  soda  (i-io  molecule)  diluted  with  about  300  cc.  of  distilled 
water.  Transfer  to  a  500  cc.  measuring  flask  and  make  the  volume  up  to 
500  cc.  with  distilled  water.  Allow  the  solution  to  stand  for  at  least  24  hours 
at  room  temperature.  The  ester  is  completely  hydrolysed  to  sodium  aceto- 
acetate. 

CH3.CO.CH2.COOC2H6  +  NaOH  =  CH3.CO.CH2.COONa  +  C2H5.OH. 

The  solution  obtained  corresponds  to  a  2  per  cent,  solution  of  aceto- 
acetic  acid. 

390.  Gerhardt's  test  for  aceto-acetic  acid.     A.    To  5  cc.  of 
the  urine  in  a  test-tube  add  ferric  chloride  solution,  drop  by  drop, 
till  no  further  precipitate  of  ferric  phosphate  is  formed.     Filter. 
To  the  nitrate  add  some  more  ferric  chloride.     A  Bordeaux-red 
colour  indicates  aceto-acetic  acid. 

NOTE. — A  similar  colour  is  given  by  a  large  number  of  substances,  such  as 
salicylic  acid,  and  the  bodies  excreted  after  the  administration  of  aspirin, 
antipyrin,  thallin,  etc.  The  majority  of  these  substances  are  not  destroyed  by 
boiling,  whereas  aceto-acetic  acid  is  converted  into  acetone.  The  reaction  is 
not  obvious  when  less  than  0-07  per  cent,  of  aceto-acetic  acid  is  present. 

*  Guy's  Hospital  Reports,  Ixvii.,  p.  161. 


CH.  XII.]  ACETO-ACETIC  ACID.  317 

B.  If  A  is  positive  shake  50  cc.  of  urine  and  3  drops  of  strong 
sulphuric  acid  with  ether.  Pipette  off  the  ether  and  treat  it  with 
very  dilute  ferric  chloride.  The  lower  layer  becomes  coloured 
violet.  Add  more  ferric  chloride.  The  colour  changes  to  a  Bordeaux- 
red. 

NOTE. — It  is  advisable  to  shake  the  acidified  urine  first  with  chloroform 
or  benzene,  to  extract  salicylic  acid. 

391.  Hartley's  test  for  aceto-acetic   acid.     To   10   cc.   of 

urine  add  2-5  cc.  of  concentrated  hydrochloric  acid  and  I  cc.  of  a 
freshly  prepared  I  per  cent,  solution  of  sodium  nitrite.  Shake  and 
allow  to  stand  for  two  minutes.  Now  add  15  cc.  of  strong  ammonia, 
then  5  cc.  of  a  10  per  cent,  solution  of  ferrous  sulphate.  Shake, 
pour  into  a  large  boiling  tube  and  allow  to  stand  undisturbed.  A 
violet  or  purple  colour  slowly  develops  if  aceto-acetic  acid  be  present. 
The  speed  at  which  the  colour  develops  depends  on  the  concentration 
of  aceto-acetic  acid.  With  small  amounts  the  colour  may  not 
develop  for  about  5  hours.  The  test  shows  in  a  dilution  of  I  in 
50,000. 

Jj.     Glycuronic  Acid. 

Glycuronic  acid,  CHO.(CHOH)4.COOH,  is  not  found 
free  in  the  urine.  It  is  found  conjugated  with  certain 
drugs,  or  with  substances  formed  from  these  in  the  body. 
These  conjugated  glycuronates  are  excreted  after  ad- 
ministration of  chloral,  camphor,  naphthol,  menthol, 
phenol,  morphine,  oil  of  turpentine,  antipyrin,  etc.  The 
free  and  conjugated  acids  are  reducing  substances,  but 
are  not  fermentable.  They  give  the  reactions  for  the 
pentoses,  but  can  be  distinguished  by  the  test  given  below. 

392.  Tollen's  test  for  glycuronates.      To  5  cc.  of  the  urine 
in  a  rather  wide  test-tube  add  -5  to  i  cc.  of  a  I  per  cent,  solution  of 
naphthoresorcin  in  alcohol  and  5  to  6  cc.  of  strong  hydrochloric  acid. 
Heat  slowly  to  boiling  point  and  keep  boiling  for  i  minute,  shaking 
the  tube  the  whole  time.     Set  the  tube  aside  for  4  minutes,  then 
cool  under  the  tap  and  shake  with  an  equal  volume  of  ether.     The 
ether  is  coloured  violet  to  red,  and  when  examined  spectroscopically 
shows  two  bands,  one  on  the  D  line,  and  one  to  the  right  of  it. 


3*8  URINE.  [CH.  XII. 

12.     Indican. 

Indican   is   the   potassium   salt   of  indoxyl  sulphuric 
acid,  and  is  thus  one  of  the  ethereal  sulphates  (seep.  281). 
Indoxyl  is 

CH 


HG  C C.OH 

HO  C          C.H 

\/\/ 

CH        NH. 

Indican  is 

CH 

/\ 

HC  C C.O.SOaK 

HC  C          CH 

\/\/ 

CH        NH. 

Indoxyl  arises  from  the  bacterial  decomposition  of 
tryptophane  in  the  intestine,  thus  differing  from  the 
other  ethereal  sulphates  which  are  normal  tissue  meta- 
bolites (see  p.  282).  The  excretion  of  indican  is  of  import- 
ance as  a  measure  of  the  amount  of  putrefaction  occurring, 
generally  in  the  intestine,  but  sometimes  in  a  large  abscess. 

393.  Jaffe's  test.  Treat  5  cc.  of  urine  with  a  rather  larger 
volume  of  concentrated  hydrochloric  acid  and  about  2  cc.  of  chloro- 
form. Add  a  single  drop  of  5  per  cent,  potassium  chlorate  and  mix. 
Allow  the  chloroform  to  settle  and  examine  its  colour.  If  it  be  blue, 
indican  is  present.  If  not,  add  another  drop  of  the  chlorate  and  mix 
again.  If  no  blue  colour  be  found  in  the  chloroform,  indican  is 
absent. 

NOTE. — The  extraction  with  chloroform  is  best  done  by  repeatedly  pour- 
ing the  mixture  from  one  tube  to  another.  It  is  essential  to  add  at  least  an 
equal  volume  of  strong  hydrochloric  acid  to  liberate  free  indoxyl.  This  is 
oxidised  to  indigo  blue,  which  is  soluble  in  chloroform. 

Horse's  and  cow's  urine  nearly  always  contain  indican.  If  procurable 
they  should  be  used  for  the  sake  of  experience. 


CH.  XII.]  UNORGANISED    SEDIMENTS.  319 

L.    Urinary  Sediments. 

For  the  proper  examination  of  these  substances  a 
hand  centrifuge  is  desirable.  The  sediment  obtained 
should  be  examined  microscopically,  and  chemically  if 
necessary. 

The  sediments  obtained  are  either  organised  or  un- 
organised. Organised  sediments  consist  of  casts  of  the 
renal  tubules,  epithelial  cells  from  different  parts  of  the 
urinary  tract,  pus,  blood  cells,  spermatozoa,  parasites,  etc. 
It  is  not  thought  advantageous  to  describe  them  in  this 
book. 

Unorganised  sediments  vary  with  the  reaction  of  the 
urine.  The  more  common  varieties  are  given  below. 

In  acid  urine. 

Uric  acid :  light  yellow  to  dark  reddish-brown  in 
colour.  Crystalline  form  very  varied  :  rhombic  prisms, 
wedges,  rosettes,  dumb-bells,  whetstones,  butchers'  trays, 
etc.  Soluble  in  sodium  hydroxide  and  reprecipitated  by 
hydrochloric  acid. 

Urates  :  pinkish,  soluble  on  warming,  sometimes 
amorphous,  sometimes  crystalline,  as  "  thorn-apples,"  fan- 
shaped  clusters  of  prismatic  needles. 

Calcium  oxalate  :  octahedra,  with  an  envelope-like 
appearance  (squares  crossed  by  two  diagonals) ;  also  in 
dumb-bells.  Insoluble  in  acetic  acid,  easily  soluble  in 
hydrochloric  acid. 

Calcium  hydrogen  phosphates  (stellar  phosphates) :  in 
rosettes  of  prisms  and  in  dumb-bells.  Rather  rare. 

Cystine  :  colourless  hexagonal  plates,  soluble  in 
ammonia,  insoluble  in  acetic  acid.  Very  rare. 


320  URINE.  [CH,  XII. 

In  alkaline  urine. 

Ammonium  magnesium  phosphate  (triple  phosphate)  : 
colourless  prisms  ("coffin-lids"  and  "knife-rests")  or 
feathery  stars.  Easily  soluble  in  acetic  acid. 

Alkaline  earthy  phosphates  of  calcium  and  magnesium  : 
amorphous.  Insoluble  on  warming  and  in  alkalies,  soluble 
in  acetic  acid. 

Calcium  hydrogen  phosphate  :  see  above. 

Calcium  carbonate  :  dumb-bells  or  spheres  with  radiat- 
ing structure. 

Ammonium  urate  :  yellow,  or  brownish  amorphous 
masses,  or  shewing  "thorn-apple"  crystals.  Soluble  on 
warming. 


CHAPTER   XIII. 
THE    QUANTITATIVE   ANALYSIS    OF    URINE. 

To  determine  the  nature  of  the  metabolic  processes 
in  the  body  a  sample  of  the  measured  24  hours'  urine 
must  be  analysed.  In  taking  the  24  hours'  urine  it  is 
best  to  finish  with  that  voided  after  the  night's  rest. 
The  total  collected  during  the  24  hours  is  mixed  and 
carefully  measured.  The  analyses  should  be  performed 
as  soon  as  possible,  owing  to  the  risk  of  bacterial  decom- 
position of  certain  of  the  constituents.  Should  it  be 
necessary  to  postpone  the  analyses  an  antiseptic  should 
be  added.  Toluol  is  the  best  to  use.  Chloroform  must 
not  be  used  in  any  case,  since  it  is  decomposed  by  alkalies 
and  has  a  marked  effect  on  certain  processes. 

The  analyses  performed  will  vary  with  the  nature 
of  the  case  that  is  being  investigated,  and  the  time  and 
apparatus  at  the  disposal  of  the  analyst.  It  is  of  the 
utmost  importance  for  the  student  to  acquire  skill  in  the 
conduction  of  a  complete  analysis,  and  it  is  to  be  hoped 
that  the  specially  selected  methods  described  below  will  be 
practised  until  satisfactory  results  can  be  obtained  in  the 
minimum  of  time.  By  careful  organisation  in  a  well- 
equipped  laboratory  it  is  possible  to  estimate  total  nitrogen, 
urea,  ammonia,  acidity,  uric  acid  and  creatinine  in  three 
hours. 

The  remarks  on  pages  380  to  382  on  the  use  of  pipettes, 
etc.,  should  be  carefully  studied  before  undertaking  any 
analytical  work.  For  the  method  of  returning  the  results 
of  analyses  see  the  form  on  page  374. 

A.    Total  Nitrogen  by  KjeldahPs  method. 

Principle.  The  nitrogenous  compounds  in  a  given  volume  of  urine  are 
converted  into  ammonium  sulphate  by  boiling  with  sulphuric  acid,  copper 
sulphate  being  added  to  aid  the  oxidation,  and  potassium  sulphate  to  raise 

X 


322 


ANALYSIS   OF   URINE. 


[CH.  XIII. 


the  boiling  point.  The  mixture  is  diluted  with  water,  made  alkaline  by  the 
addition  of  sodium  hydroxide  and  the  ammonia  distilled  into  a  measured 
amount  of  standard  acid.  The  amount  of  this  neutralised  by  the  ammonia  is 
found  by  subsequent  titration  with  standard  alkali.  Knowing  the  amount 
of  ammonia  formed  from  the  volume  of  urine  taken,  the  percentage  of  nitrogen 
can  be  readily  calculated. 

The  technique  adopted  varies  with  the  material  and  with  the  inclinations 
of  the  operator.  There  are  four  main  methods  of  distillation,  viz.,  by  direct 
boiling,  by  steam,  by  boiling  with  alcohol,  and  by  the  author's  method  of 
combined  boiling  with  alcohol  and  aeration. 

The  main  objection  to  direct  boiling  is  that  the  mixture  is  apt  to  bump 
very  violently,  and  that  a  certain  loss  of  ammonia  may  occur  when  adding  the 
alkali.  The  bumping  is  much  less  with  potash  than  with  soda,  but  the  price 
is  prohibitive. 

Steam  distillation  is  much  safer,  and,  although  it  is  slow,  it  needs  very 
little  attention.  It  is  the  best  method  when  considerable  amounts  of  sulphuric 
acid  have  to  be  used  for  the  incineration. 

Distillation  with  alcohol  is  very  smooth  and  relatively  rapid,  for  the 
alcohol  boils  quickly  and  carries  the  ammonia  over  with  it. 

The  combination  of  aeration  with  alcohol  distillation  (Ex.  397)  was 
originally  devised  for  the  estimation  of  very  small  amounts  of  nitrogen,  but 
it  is  so  rapid  and  accurate  that  the  author  now  uses  it  for  the  estimation  of  total 
nitrogen  in  urine.  Either  0-5  or  i  cc.  of  the  urine  is  measured  with  an  Ostwald 
pipette,  the  incineration  is  completed  in  15  mins.,  and  the  result  is  obtained  in 
less  than  30  mins.  The  results  agree  exactly  with  those  obtained  when  5  cc. 
of  urine  are  taken. 

Standard  acids  and  alkalies  and  indicators.  It  is  essential  to  use  CO2-free 
soda  for  the  back  titrations.  The  end  point  is  extremely  sharp  if  methyl  red 

is  used  as  an  indicator.  With 
ordinary  soda  the  yellow  tint 
obtained  when  the  solution  is 
just  alkaline  soon  changes  to 
a  pink,  and  there  is  great  un- 
certainty as  to  how  far  to  pro- 
ceed with  the  titration.  This 
is  fatal  to  good  results.  With 
CO2-  free  soda,  on  the  other 
hand,  the  change  of  tint  is 
brought  about  by  the  addition 
of  less  than  a  drop  of  0*02  N. 
soda,  and  is  permanent  for  a 
considerable  time.  The  titra- 
tion can  be  conducted  by  arti- 

A...,.  ficial  light,   which   is  not  the 

case  with  methyl  orange.  The 
preparation  of  CO2-  free  soda 
is  described  on  page  26.  The 
strength  required  varies  with 
the  method  adopted.  For 
ordinary  work  the  author  pre- 
fers to  use  about  o-i  N. 
strength,  but  for  the  micro- 
method  it  is  better  to  use  0-03  to  0-05  N.  As  a  rough  guide  it  may  be 
stated  that  the  strong  soda  prepared  according  to  the  directions  on  page  25 


B. 


Fig    39- 

Hall's  two-way  tube 
for  burettes. 


CH.  XIII.]  TOTAL   NITROGEN.  323 

is  about  10  N.  A  paraffined  bottle,  a  grease-free  burette  (see  p.  381)  and  a  good 
supply  of  recently  boiled  and  cooled  distilled  water  being  ready,  the  bottle  is 
nearly  filled  with  the  water,  the  volume  of  which  is  noted.  The  requisite 
amount  of  the  strong  CO2-  free  soda  is  added,  the  solution  well  mixed,  and  the 
burette  and  soda-lime  tubes  immediately  fitted.  It  is  advisable  to  seal  all  the 
joints  with  paraffin  wax,  to  prevent  the  absorption  of  atmospheric  CO2,  and 
to  ensure  the  due  filling  of  the  burette  by  suction.  It  is  difficult  to  fit  the 
upper  stopper  to  an  ordinary  burette.  To  overcome  this,  Messrs.  Baird  and 
Tatlock  are  making  burettes  fitted  with  a  wide  top,  similar  to  that  shewn  on 
page  130.  Mr.  H.  W.  Hall,  of  the  Cambridge  Biochemical  Laboratory,  has 
made  it  possible  to  use  an  ordinary  burette  by  the  construction  of  the  two-way 
piece  shewn  in  fig.  39. 

The  exact  concentration  of  the  soda  is  determined  by  titration  of  a 
weighed  amount  of  acid  potassium  phthalate,  or  by  the  use  of  an  acid  of 
known  normality. 

The  acid  employed  can  be  hydrochloric  or  sulphuric.  In  the  case  of  the 
aeration  methods  it  is  advisable  to  use  the  latter,  to  avoid  any  risk  of  loss  by 
volatility.  The  strength  should  be  somewhat  greater  than  that  of  the  alkali. 
A  great  deal  of  time  is  saved  if  the  acid  be  measured  from  a  burette  rather  than 
with  a  pipette.  The  initial  reading  is  noted,  the  approximate  amount  required 
is  run  in,  and  the  burette  allowed  to  stand  until  the  final  titration  is  performed. 
The  soda  is  then  run  in  until  the  solution  goes  yellow,  a  little  acid  is  added  until 
a  pink  tint  appears.  The  sides  of  the  vessel  are  washed  down  with  a  little 
distilled  water,  and  the  titration  completed  by  the  addition  of  a  few  drops  of 
the  soda.  In  this  way,  being  relieved  from  the  anxiety  that  one  may  overshoot 
the  mark  with  the  alkali,  it  is  not  necessary  to-  run  the  whole  of  the  alkali  in 
drop  by  drop,  which  may  take  a  considerable  time.  The  only  danger  is  that 
of  allowing  insufficient  time  for  proper  drainage  of  the  alkali  burette. 

Calculation. 
i  gram. -molecule  of  acid  neutralises  i  gram. -molecule  of  ammonia. 

So  1,000  cc.  of  N.HC1  (or  other  acid)  neutralises  17  grams.  NH3  and  are 
equivalent  to  14  grams,  of  Nitrogen. 

So  i  cc.  of  N.  acid  =  14  mgms.  Nitrogen. 

So  i  cc.  of  (A)  x  N.  acid  =  14  x  (A)  mgms.  Nitrogen  ("  acid  equivalent.") 

Suppose  that  the  soda  employed  be  (S)  normal, 

(S) 

Then  i  cc.  soda  =  TTT  cc.  acid  ("  alkali-acid  ratio  "). 
(A) 

Let  the  amount  of  acid  used  be  (a)  cc.,  and  the  amount  of  alkali  be  (s)  ce. 

(S)  x  (s) 

Then  (5)  cc.  alkali  =  — T-TT —  cc.  acid. 
(A) 

So  volume  of  acid  neutralised  by  the  ammonia  distilled  over  is 

(S)  x  (5)' 
(a)  -  — /Av  '     cc.,  and  the  amount  of  nitrogen  is 

I  W  ~  — (Af" J  x  T4  x  (A)  mg- 
A  good  deal  of  time  is  saved  if  the  acid  and  alkali  be  labelled  with  the 


324  ANALYSIS    OF    URINE.  [CH.  XIII. 

logarithms  of  the  "  acid-equivalent  "  (acid  log.)  and  of  the  "  alkali-acid  ratio  " 
(alkali  log.)  respectively. 

The  following  example  should  be  carefully  studied. 
Acid  employed  was  0-0476  N.  sulphuric  (acid  log.      =  1-8237). 
Alkali  employed  was  0-0421  N.  soda  (alkali  log.  =  1-9467). 

0-5  cc.  of  urine  taken. 

Amount  of  acid  taken  was  20-12  cc.,  and  this  required  13-6  cc.  of  alkali 
for  back  titration. 

Log.  of  13-6  =  Jf-1335 

Add  the  alkali  log  i  -9467 


Anti-log,  of          1-0802  is  12-03. 

So  20- 12  -  12-03  =  8-09  cc.  of  acid  have  been  neutralised  by  the 
ammonia  formed  from  0^5  cc.  of  urine. 

Log.  of  8-09  is  '9079 

Add  the  acid  log.  i  -8237 

Anti-log,  of  -7316  is  1-539- 

So  0-5  cc.  of  urine  contain  1-539  mg.  total  Nitrogen. 
So  100  cc.  of  urine  contain  0-308  gram,  total  Nitrogen. 

(This  example  is  from  an  analysis  of  a  very  dilute  urine.  The  figure  is 
usually  about  i  per  cent.) 

No  allowance  has  been  made  for  the  blank  determination,  but  this  should 
not  be  neglected,  especially  when  using  the  micro-method.  The  blank 
determination  is  made  with  all  the  materials  used  for  an  ordinary  analysis, 
distilled  water  being  take  instead  of  urine.  Unless  the  reagents  are  of  very 
poor  quality,  the  amount  of  nitrogen  found  should  be  very  small.  This  must 
be  deducted  from  the  amount  found  in  the  volume  of  urine  taken.  An 
example  is  given  on  page  263. 

394.  Kjeldahl's  method  (distillation  by  boiling).  Into  a 
clean,  dry,  round-bottomed  flask  of  "  Duro  "  glass  A  (500  cc. 
capacity,  with  a  narrow  neck  8  inches  in  length)  place  5  to  10 
grams,  potassium  sulphate,  0-5  cc.  of  saturated  copper  sulphate 
solution,  5  cc.  of  urine  (accurately  measured)  and  10  cc.  of  concen- 
trated sulphuric  acid,  free  from  nitrogen.  Place  the  flask  in  the 
fume-chamber  (or  use  the  fume-absorber,  described  on  page  383), 
and  heat  by  means  of  a  low  flame  for  10-15  minutes,  then  boil  briskly 
for  45  minutes  or  longer.  The  solution  must  be  heated  for  at  least 
15  minutes  after  it  has  lost  every  trace  of  dark  colour.  Any  particles 
of  carbonaceous  matter  that  adhere  to  the  sides  of  the  flask  must  be 
washed  down  into  the  acid  by  carefully  shaking  the  flask.  When 
cool*:  add  250  cc.  of  ammonia-free  distilled  water,  3  or  4  pieces  of 
broken  porous  pot,  and  cool  under  the  tap.  Into  an  Erlenmeyer 


CH.  XIII.] 


TOTAL   NITROGEN, 


325 


flask,   E,   of   about   400   cc.   capacity,   place  20  cc.    of   standard 
sulphuric  acid,  about  0*2  N. 

This  flask  is  then  placed  on  an  ad- 
justable stand,  so  arranged  that  the  lower 
end  of  the  tube  D  dips  below  the  surface 
of  the  acid  in  E.  The  bulb  in  D  is  to 
decrease  the  risk  of  the  acid  in  E  being 
sucked  back  by  a  sudden  cooling  of  A 
during  the  distillation.  D  is  connected 
to  a  condenser  C.  The  best  pattern  is 
Davies',  which  is  shewn  in  fig.  40. 

To  the  flask  A  add  35  to  40  cc.  of 
40  per  cent,  sodium  hydroxide,  pouring 
it  down  the  neck  and  wall  of  the  flask 
so  as  to  form  a  bottom  layer  ;  loss  of 
ammonia  is  thus  prevented. 

Fit  the  glass  tube  B  into  the  neck 
of  A  by  means  of  a  well-fitting  rubber 
stopper.  The  special  bulb  on  B  is  to 
prevent  any  of  the  alkaline  fluid  bumping 
over  into  the  distillate. 

Mix  the  contents  of  A  by  shaking 
and  immediately  connect  up  B  with  C 
by  means  of  another  well-fitting  rubber 
stopper.  Heat  the  mixture  in  A  to  boil- 
ing by  means  of  a  free  flame  from  a 
Bunsen  burner  provided  with  a  rose-top. 
Allow  the  fluid  to  boil  till  at  least  half 
the  total  volume  of  fluid  has  distilled 
over,  lowering  E  from  time  to  time,  so  Fig-  4°-  Kjeidahl  appara- 
that  D  does  not  dip  too  far  under  the  £rsect  bS^*' 
acid.  Finally,  lower  E  so  that  the  tube 

no  longer  dips  under  the  surface  and  continue  the  boiling  for 
another  minute  or  two  to  wash  down  any  of  the  standard  acid 
that  may  have  been  sucked  up  into  the  tube  or  bulb.  Wash 
down  the  exterior  of  the  lower  end  of  D  with  a  jet  of  distilled  water, 
allowing  the  washings  to  run  into  E. 


326  ANALYSIS   OF   URINE.  [CH.  XIII. 

To  the  fluid  thus  obtained  add  a  few  drops  of  a  0-02  per  cent, 
solution  of  methyl  red  and  titrate  with  standard  CO2-  free  sodium 
hydroxide,  which  may  be  between  o-i  and  0-15  N. 

Calculation,  see  p.  323. 

395.  Kjeldahl's  method  (steam  distillation).  The  incinera- 
tion is  conducted  as  described  in  the  previous  exercise,  but  a 
smaller  Kjeldahl  flask  may  be  used  if  desired.  After  the  fluid 


Fig.  41.     Kjeldahl  apparatus.     Steam  distillation. 

has  cooled,  add  50  cc.  of  distilled  water,  shake  round  well  and 
transfer  the  solution  to  D,  a  round-bottom  flask  of  i  or  2  litres, 
with  a  neck  sufficiently  wide  to  carry  a  well-fitting  rubber  stopper 
that  will  allow  3  tubes  to  pass  through,  as  shewn  in  the  figure. 
Wash  out  the  Kjeldahl  flask  twice  more,  using  about  25  cc.  of 
distilled  water  each  time.  Measure  the  requisite  amount  of  standard 
acid  into  K,  and  assemble  the  apparatus.  Arrange  the  wooden 
blocks,  L,  so  that  the  end  of  the  delivery  tube,  H,  just  dips  under 
the  surface  of  the  acid.  (It  is  incorrectly  drawn  in  the  figure.)  E  is 
a  tap-funnel  containing  40  per  cent.  soda.  G  is  a  condenser,  the 
double-surface  variety  being  the  most  efficient. 


CH.  XIII.]  TOTAL   NITROGEN.  327 

The  water  in  the  copper  vessel  A  has  previously  been  vigorously 
boiled  for  at  least  ten  minutes  to  ensure  the  removal  of  any  ammonia. 
Remove  the  flame  for  a  moment  and  connect  the  exit  pipe  of  the 
boiler  to  C  by  rubber  tubing.  Replace  the  burner.  Run  in  the 
strong  soda  from  E  until  the  solution  is  definitely  alkaline,  as  can 
be  seen  by  the  fluid  turning  blue,  due  to  the  formation  of  cupric 
hydroxide.  The  distillation  must  be  allowed  to  proceed  for  at 
least  45  minutes.  It  is  safer  to  allow  an  extra  half-hour.  The  only 
attention  necessary  is  to  see  that  there  is  a  sufficient  amount  of 
water  in  the  boiler  and  that  the  flask  K  is  at  the  right  height. 

At  the  end  of  the  operation,  remove  the  blocks  from  under  K, 
so  that  H  does  not  dip  into  the  acid.  After  a  few  minutes,  remove 
the  flame,  wash  down  the  interior  and  exterior  of  H  into  K,  and 
titrate  as  described  on  p.  323. 

Calculation,  see  p.  323. 

396.  Kjeldahl's  method  (alcohol  distillation).  Into  a  500  cc. 
Kjeldahl  flask  of  "  Duro  "  glass  measure  2  cc.  of  the  urine,  using  an 
Ostwald  pipette  (fig.  48).  Add  3  cc.  of  pure  concentrated  sulphuric 
acid,  2  grams,  of  potassium  sulphate  and  2  drops  of  saturated 
copper  sulphate  solution.  Heat  over  a  micro-burner,  using  a  Folin's 
fume-absorber.  The  flame  should  be  about  half  an  inch  in  height, 
and  should  play  directly  on  the  bottom  of  the  flask  to  ensure  boiling. 
Any  particles  of  carbonaceous  matter  that  form  on  the  side  of  the 
flask  must  be  rinsed  down  into  the  acid.  The  heating  must  be 
continued  for  5  to  10  minutes  after  the  solution  has  turned  blue. 
Remove  the  flame  and  allow  the  solution  to  cool  until  the  flask  is 
only  pleasantly  warm  to  the  hand.  Add  20  cc.  of  distilled  water 
from  a  measuring  cylinder.  This  should  be  added  rapidly,  and  the 
mixture  immediately  shaken  to  prevent  the  formation  of  a  cake  of 
potassium  hydrogen  sulphate.  Cool  under  the  tap.  Add  three  or 
four  pieces  of  broken  porous  pot  and  15  cc.  of  95  per  cent,  alcohol. 
Assemble  the  apparatus,  seeing  that  the  clamps  are  correctly 
adjusted,  so  that  the  rubber  stoppers  fit  into  the  flasks  without 
undue  strain.  G  is  a  piece  of  glass  rod,  with  the  lower  end  flattened 
out  and  bent  up  as  shewn.  It  is  passed  up  through  the  rubber 
stopper,  and  the  upper  end  can  then  be  flattened  out,  if  desired,  for 
convenience  of  manipulation.  The  tube  is  drawn  up  until  the  flange 


328 


ANALYSIS    OF    URINE. 


[CH.  XIII. 


is  about  i  inch  from  the  exit  tube  B.  This  minimises  the  risk  of  any 
alkali  being  carried  over  by  spurting.  This  risk,  however,  has  been 

found  to  be  so  small  that  it  is  hardly 
worth  the  trouble  of  fitting.  B  is 
joined  to  the  tube  to  the  condenser 
by  a  short  piece  of  rubber  tubing. 
This  allows  the  operator  to  shake  A, 
and,  by  removing  the  strain,  de- 
creases the  risk  of  breaking  the 
glass  parts.  The  standard  acid  is 
measured  into  E,  a  250  cc.  Erlen- 
meyer  flask.  20  cc.  of  o-i  N.  acid 
is  usually  ample.  The  wooden 
blocks,  F,  should  be  so  arranged 
that  the  lower  end  of  D  only  just 
dips  below  the  surface  of  the  acid. 
Now  remove  A  and  run  in  13  or 
14  cc.  of  40  per  cent,  soda,  running 
this  gently  down  the  lower  part  of 
the  neck  and  sides  of  the  flask,  so 
that  the  soda  sinks  to  the  bottom 
of  the  fluid  and  the  risk  of  the  loss  of 
ammonia  is  minimised.  The  flask 
should  be  held  in  a  nearly  horizontal 
position  during  this  operation;  it 
should  be  raised  to  the  vertical  caut- 
iously, so  as  to  prevent  mixing  of  the 
two  layers.  Re-assemble  the  appa- 
ratus, seeing  that  the  two  rubber 

stoppers  are  firmly  held.  Have  ready  a  Bunsen  burner,  with  a 
rose-top,  and  a  screw  clip,  H,  fitted  to  the  rubber  tubing.  See 
that  the  adjustable  stand  for  the  burner  is  at  such  a  height  that  the 
top  of  the  rose  is  about  half  an  inch  under  the  bottom  of  the  flask. 
Turn  on  the  water  supply  to  the  condenser.  Unclamp  the  flask  A 
and  mix  its  contents  by  shaking,  taking  care  that  the  rubber  stoppers 
are  not  loosened.  Place  the  burner  in  position  and  gently  agitate 
the  flask  until  the  fluid  commences  to  boil.  The  flask  can  then  be 
clamped  and  the  distillation  allowed  to  continue  for  15  minutes. 


Fig.  42.      Kjeldahl's  method. 

Cole's  apparatus  for  alcohol 
distillation. 


CH.    XIII.]  MICRO-KJELDAHL.  329 

Remove  the  wooden  blocks  F,  and  then  remove  the  flame.  Remove 
the  rubber  stopper  from  the  upper  end  of  the  condenser  and  wash 
the  latter  down  into  E  with  a  jet  of  distilled  water.  Wash  down 
the  exterior  of  D  also,  add  a  few  drops  of  methyl  red  and  titrate 
with  the  CO2-  free  soda,  as  described  on  page  323.  The  soda  can  be 
between  0-05  and  o-i  N. 

Calculation,  see  p.  323. 

397.  Micro-Kjeldahl  (Cole's  method).  Into  a  clean,  dry, 
boiling  tube  measure  0-5  or  I  cc.  of  the  urine,  using  an  Ostwald 
pipette  (fig.  48).  Add  2  cc.  of  pure  concentrated  sulphuric  acid  and 
2  drops  of  saturated  copper  sulphate.  Mix  and  boil  over  a  free 
flame,  shaking  vigorously  to  prevent  spurting,  until  dense  white 
fumes  appear  in  the  tube.  A  test-tube  holder  should  be  improvised 
by  the  use  of  a  piece  of  folded  paper.  Add  i  gram,  of  pure  potassium 
sulphate  and  heat  again.  Clamp  the  tube  over  a  micro-burner, 
having  a  flame  about  J  inch  in  height,  just  touching  the  bottom  of 
the  tube,  and  insert  a  Folin's  fume-absorber  (fig.  51)  into  the  mouth 
of  the  tube.  Continue  the  gentle  boiling  for  at  least  5  minutes  after 
the  solution  has  lost  all  trace  of  its  dark  colour  and  has  turned  light 
blue.  Allow  to  cool,  add  10  cc.  of  distilled  water,  transfer  the 
solution  to  the  tube  B  shewn  in  fig.  34,  p.  261,  and  proceed 
exactly  as  described  in  the  last  paragraph  of  Ex.  311. 

B.    The  Estimation  of  Ammonia. 

The  ammonia  of  urine  normally  exists  as  ammonium  salts  of  weak  acids . 
In  cases  of  cystitis,  however,  the  urine  is  nearly  always  alkaline,  owing  to  the 
conversion  of  some  of  the  urea  to  ammonium  carbonate  by  various  micro- 
organisms. This  change  may  occur  after  the  urine  has  been  passed  owing 
to  bacterial  contamination  from  the  air,  etc.  For  this  reason  it  is  essential 
that  a  little  toluol  should  be  added  to  the  vessel  in  which  the  urine  of  the 
24  hours  is  being  collected,  that  it  should  be  kept  in  a  cool  place  and  that  the 
estimations  should  be  made  as  soon  as  possible. 

A  great  many  methods  have  been  proposed  for  the  estimation  of  ammonia 
in  urine.  Folin  has  introduced  'them  at  a  rate  which  is  almost  alarming. 
Indeed,  one  might  be  led  to  imagine  that  such  a  master  of  technique  distrusts 
his  own  methods  so  much  that  he  is  forced  to  strive  for  a  better  one.  Nearly 
all  his  later  methods  are  colorimetric,  a  most  excellent  modification  of  Nessler's 
solution  having  been  elaborated.  But  the  author's  experience  with  large 
classes  is  that  the  majority  of  workers  prefer  to  use  a  titration  method  if 
possible.  Mainly  for  that  reason,  the  only  three  methods  described  here  are 
Folin's  original  macro-method,  Van  Slyke's  modification  of  it,  and  the  formol 
method.  The  latter,  however,  gives  the  sum  of  ammonia  and  the  amino-acids, 


330 


ANALYSIS    OF    URINE. 


[CH.  XIII. 


and  the  results  obtained  by  it  are  only  of  approximate  value  for  the  ammon'a 
figure.     It  will  be  described  in  connexion  with  the  estimation  of  amino-acids. 

Of  the  two  methods  described  below,  the  author  now  always  employs  Van 
Slyke's,  which  is  much  more  rapid  than  Folin's.  The  author  is  convinced 
that  failures  to  obtain  correct  results  are  either  due  to  inattention  to  essential 
details  or  to  the  use  of  an  imperfect  suction  pump.  An  apparatus  that  sup- 
plies air  under  a  good  pressure  is  a  most  valuable  adjunct  to  a  modern  bio- 
chemical laboratory. 

398.    The  estimation  of  ammonia  by  Folin's  method. 


, — U-[LTO  PUN,. 


Fig.  43.     Folin's  apparatus  for  estimating  ammonia. 

A.  Wash  bottle  containing  acid. 

B.  Tall  aerometer  cylinder  containing  urine. 

C.  Bottle  containing  standard  acid. 

D.  Calcium  chloride  tube,  loosely  packed  with  cotton  wool,  to  prevent 

any  sodium  carbonate  being  carried  over  into  C. 

E.  Folin's  absorption  tube,  to  bring  the  air  into  intimate  contact  with 

the  acid. 

Use  the  apparatus  shewn  above.* 


*  The  parts  of  the  apparatus  can  be  obtained  from  Messrs.  Baird  and 
Tatlock  (London). 


CH.   XIII.] 


AMMONIA. 


331 


Into  C  measure  20  cc.  of  standard  sulphuric  acid  (about  o-i  N.) 
and  a  few  drops  of  methyl  red. 

Into  B  measure  25  cc.  of  urine,  add  5  or  6  drops  of  caprylic 
alcohol  (to  prevent  foaming)  and  2  grams,  of  anhydrous  sodium 
carbonate.  Connect  up  the  apparatus  at  once,  and  draw  air  through 
for  two  hours. 

Disconnect  the  apparatus,  wash  the  tube  E  with  distilled  water 
into  C,  and  titrate  with  CO2-  free  sodium  hydroxide  (about  o-i  N.). 

Calculation.  Determine  the  percentage  of  nitrogen  in  the  form  of 
ammonia  as  described  for  Kjeldahl's  method,  p.  323.  The  result  thus  obtained 
is  the  mgms.  of  ammonia-nitrogen  in  25  cc. 

To  convert  this  to  grams,  of  ammonia  per  cent.  ,  multiply  by 

4  x         x  =  °'°°486  (l°g-  3-6864). 


To  find  the  ammonia  in  terms  of  cc.  of  o-i  N.  acid  per  cent.,  multiply  the 

10 
mgms.  of  ammonia-nitrogen  in  25  cc.  by  4  x  —  =  2-86  (log.  '4560)- 

399.    Van  Slyke's  method. 

Principle.  5  cc.  of  urine  are  made  strongly  alkaline  with  potassium 
carbonate,  which  decomposes  the  ammonium  salts.  The  ammonia  liberated 
is  driven  over  by  an  air  current  into  a  measured  amount  of  standard  acid, 
which  is  subsequently  titrated  with  standard  alkali.  The  treatment  of  urine 
at  room  temperature  with  potassium  carbonate  does  not  lead  to  the  formation 
of  ammonia  from  urea,  etc.,  as  does  boiling  with  caustic  soda. 

Apparatus.     This  is  shewn  in  fig.  44.*     A  is  a  wash  bottle  containing 


To  pump 


C-- 


F..-.\ 

Fig.  44.     Apparatus  for  estimation  ol  ammonia  and  urea  by  Van  Slyke's 

methods. 

*  This  can  be  obtained  from  Messrs.  Baird  and  Tatlock  (Ltd.),  14,  Cross 
Street,  Hatton  Garden,  London,  E.G. 


332  ANALYSIS    OF    URINE.  [CH.   XIII. 

sulphuric  acid  (i  in  10)  to  remove  ammonia  from  the  air.  B  is  a  large  thick- 
walled  tube,  25  to  30  mm.  by  200  mm.  C  is  a  sheet  of  rubber,  about  2  mm. 
thick,  cut  from  a  2-holed  rubber  stopper.  It  fits  loosely  into  B,  and  has  a 
small  groove  cut  at  the  side.  It  decreases  the  risk  of  an  alkali  foam  being 
carried  over  into  E,  which  is  a  tube  similar  to  B.  D  can  be 
made  from  a  broken  5  cc.  pipette  and  may  be  loosely  filled 
with  cotton  wool.  F  is  a  tube  sealed  at  the  lower  end  with 
holes  bored  in  it,  whilst  still  hot,  with  a  hot  needle.  The  tubes 
B  and  E  are  conveniently  held  by  means  of  a  heavy  wooden 
block  bored  with  two  large  holes.  In  place  of  the  tube  E,  a 
100  cc.  flask  with  a  wide  neck  may  be  substituted  as  shewn  in 
fig.  45.  The  advantage  of  the  flask  is  that  there  is  very  little 
risk  of  the  standard  acid  being  carried  over  with  the  brisk  air 
current  necessary.  The  objection  to  it  is  that  the  depth  of 
the  acid  layer  being  decreased  there  may  be  a  danger  of  loss 
of  ammonia.  If  the  air  current  is  a  moderate  one  for  the  first 
two  minutes  this  risk  is  very  slight,  and  perfect  results  are 
obtained. 

An    efficient    suction    pump    or    blast     pump    is    also 
required. 
45- 

Method,  (i.)  Into  B  measure  5  cc.  of  the  urine,  and  add  2 
drops  of  caprylic  alcohol  to  stop  foaming.  4  or  5  drops  of  kerosene 
can  be  used,  but  it  is  not  an  efficient  substitute. 

(ii.)  Into  E  measure  20  cc.  of  the  standard  sulphuric  acid, 
which  should  be  between  0-04  and  0-07  N.  If  a  flask  is  used  it  is 
advisable  to  add  about  20  cc.  of  distilled  water  to  give  a  deeper 
layer  for  absorption. 

(iii.)  Place  4  to  5  grams,  of  pure  dry  potassium  carbonate  into  B, 
roughly  measuring  it  with  a  suitable  spoon.  Immediately  connect 
up  the  apparatus,  taking  care  that  D  is  joined  to  F,  and  not  to  the 
connexion  for  the  pump.  Turn  on  the  water  supply  to  the  pump 
so  that  a  rather  slow  air  current  is  drawn  through.  After  2  to  3 
minutes,  turn  on  the  water  to  full  pressure  and  leave  it  for  12  more 
minutes. 

(iv.)  Gradually  stop  the  pump  and  disconnect  the  tube  E. 
Lift  up  the  rubber  stopper  so  that  F  does  not  dip  into  the  acid,  and 
wash  down  the  interior  of  F  with  distilled  water,  using  a  fine  jet. 
Repeat  this  twice,  allowing  time  for  proper  drainage.  Carefully 
wash  down  the  exterior  of  F  with  distilled  water,  add  3  or  4  drops 
of  methyl  red  and  titrate  with  CO2-  free  soda,  which  may  be  between 
0-03  and  0-06  N.,  according  to  the  directions  given  on  page  323. 

Calculation.    Determine  the  percentage  of  nitrogen  in  the  form  of  ammonia 


CH.  XIII.]  AMMONIA   AND   AMINO-ACIDS.  333 

as  described  for  Kjeldahl's  method,  p.  323.     The  result  thus  obtained  is  the 
mgms.  of  ammonia-nitrogen  in  5  cc.  =  A. 

Mgms.  of  ammonia-nitrogen  in   100  cc.  =  20  A. 
Grams,  of  ammonia-nitrogen  in  100  cc.  =  A  x  0-02. 

Grams,  of  ammonia  in  i  oo  cc.  =  A  x  0-02  x  —  =  A  x  0-0243  (log.  2-3853). 

cc.  of  o-i  N.  acid  neutralised  by  NH3  of  100  cc.  =  20  A  x  —  =  A  x  14-29 
(log.  1-1549)- 

400.  C.  The  estimation  of  ammonia  and  amino-acids  by 
formol  titration  (Cole's  method). 

Principle.  Neutral  ammonium  salts  react  with  an  excess  of  neutral 
formaldehyde  to  give  hexamethylene  tetramine,  the  acid  being  liberated. 

4  NH4C1  +  6  CH2O  =  N4  (CH2)6  +  6  H2O  +  4  HC1. 

From  the  amount  of  alkali  required  to  again  make  the  solution  neutral, 
the  amount  of  ammonia  can  be  estimated. 

Neutral  amino-acids  also  react  with  formol  to  give  methylene  amino-acids 
[see  p.  69  (3)  ].  The  result  of  the  estimation  therefore  gives  the  sum  of  the 
ammonia  and  the  amino-acids  of  the  urine. 

The  method  usually  adopted  is  to  neutralise  the  urine  to  phenol-phthalein, 
to  add  neutralised  formol,  which  makes  the  fluid  acid,  and  then  to  determine 
how  much  standard  soda  is  again  required  to  neutralise  the  mixture.  The 
great  difficulty  encountered  is  that  of  determining  the  neutral  point,  and 
experience  with  large  classes  of  students  has  revealed  the  fact  that  considerable 
variations  in  results  are  found  due  to  the  indecision  about  the  two  end  points. 

As  explained  on  p.  215,  the  author  has  overcome  this  difficulty  by  the  use 
of  the  comparator  shewn  in  fig.  27.  The  results  obtained  by  untrained 
students  now  agree  very  closely. 

Method.  Use  the  comparator  for  large  tubes  described  on 
p.  276. 

Into  tubes  (2),  (3)  and  (6)  measure  20  cc.  of  the  urine. 

Into  tube  (i)  measure  20  cc.  of  buffer  solution  PH  —  8*4  (see 


Into  tube  (5)  measure  20  cc.  of  buffer  solution  PH  —  8-5.* 
Into  tube  (4)  place  about  30  cc.  of  water. 

To  tubes  (i),  (3)  and  (5)  add  10  to  20  drops  of  0-5  per  cent. 
phenol  phthalein,  adding  exactly  the  same*amount  to  each  by  the 
use  of  a  dropping  pipette  (fig.  5).  The  amount  necessary  varies 

*  If  only  one  buffer  solution  is  used,  as  is  done  in  the  exercise  on  p.  215, 
it  should  be  PH  =  8-45. 


334  ANALYSIS   OF  URINE.  [CH.  XIII. 

with  the  appearance  of  the  urine,  more  being  required  for  deeply 
pigmented  urines. 

Titrate  with  standard  soda,  which  may  be  o-i  to  0-2  N.,  as 
described  in  Ex.  322,  until  the  colour  as  seen  through  Y  is  inter- 
mediate between  that  seen  through  X  and  Z.  During  the  course 
of  this  titration,  the  standard  soda  is  added  to  (2)  and  (6)  and 
distilled  water  to  (i)  and  (5),  as  described  in  Ex.  322.  Usually  a 
considerable  precipitate  of  earthy  phosphates  appears  in  the  three 
tubes  that  contain  urine.  The  contents  of  these  tubes  must  be  well 
mixed  by  rotation  or  otherwise  immediately  before  an  observation 
is  made. 

Measure  5  cc.  of  commercial  formaldehyde  (40  per  cent.)  into  a 
test-tube.  Add  one-third  the  number  of  drops  of  phenol  phthalein 
added  to  the  urine  and  then  the  standard  soda,  drop  by  drop,  until  a 
faint  pink  tinge  is  obtained.  Add  the  whole  of  this  solution  to 
tube  (3).  Note  that  the  pink  tinge  and  the  precipitate  of  earthy 
phosphates  disappear,  owing  to  the  acidity  developed.  To  tubes 
(2)  and  (6)  add  5  cc.  of  water,  to  dilute  the  urinary  pigment  to  the 
same  degree  as  that  in  tube  (3). 

Read  the  burette  containing  the  standard  soda. 

Titrate  the  contents  of  tube  (3)  with  the  soda,  until  the  appear- 
ance at  Y  approaches  that  seen  at  X.  To  tubes  (i),  (2),  (5)  and  (6) 
add  the  same  volume  of  distilled  water  as  the  soda  added  in  this  last 
operation.  Mix  the  contents  carefully  and  complete  the  titration, 
so  that  the  appearance  at  Y  is  intermediate  between  that  seen  at 
XandZ. 

Calculation.  If  (a)  cc.  of  soda  of  normality  (n)  are  required  to  neutralise 
20  cc.  after  the  addition  of  the  formol,  then  20  cc.  urine  contain  (a)  x  (n)  x  (14) 
mgms.  of  Nitrogen  of  ammonia  and  amino-acids.  So  100  cc.  urine  contain 
(a)  x  (n)  x  (70)  mgms.  of  (ammonia  +  ammo-acid)  Nitrogen.  This  amount, 
less  20  A  (the  mgms.  of  ammonia-Nitrogen  determined  in  the  previous  exercise) 
is  the  mgms.  of  amino-acid  Nitrogen  in  100  cc.  urine. 

D.     The  Estimation  of  Urea. 

The  use  of  the  enzyme  urease  (see  p.  287)  has  rendered  obsolete  a  large 
number  of  methods  that  had  been  devised  for  the  estimation  of  urea  in  urine. 
The  time  required  for  D.  Van  Slyke's  method  is  not  much  greater  than  that 
for  the  old  hypobromite  method,  and  the  results  obtained  are  accurate, 
whereas  with  hypobromite  they  are  most  unreliable. 


CH.  XIII.]  UREA.  335 

401.    Van  Slyke's  method  for  urea. 

Principle.  A  small  volume  of  the  urine  is  treated  -with  an  extract  of  the 
Soya  bean,  together  with  a  certain  amount  of  acid  potassium  phosphate  to 
preserve  the  optimum  reaction  for  the  enzyme.  The  whole  of  the  urea  is 
rapidly  converted  to  ammonium  carbonate.  An  excess  of  potassium  carbonate 
is  added,  and  the  ammonia  formed  from  the  urea,  together  with  that  from  the 
preformed  ammonium  salts  of  the  urine  are  driven  over  into  standard  acid  and 
estimated  in  the  way  described  in  Ex.  399.  The  amount  of  ammonia  being 
known,  the  percentage  of  urea  can  be  found  by  difference. 

Apparatus.  This  is  exactly  similar  to  that  required  for  Ex.  399.  A 
duplicate  set  should  be  obtained  so  that  the  ammonia  and  urea  determinations 
can  be  conducted  simultaneously. 

Urease  solution.  An  active  extract  of  the  finely  ground  Soya  beans*  can 
be  prepared  by  the  following  method.  Rub  up  5  grams,  of  the  meal  with 
50  cc.  of  0-6  per  cent,  acid  potassium  phosphate  (KH2PO4)  in  a  mortar. 
After  standing  for  about  15  minutes,  filter  through  a  pleated  paper.  The 
opalescent  solution  obtained  should  be  freshly  prepared,  but  if  kept  in  an  ice 
chest  it  seems  to  be  stable  for  2  or  3  days. 

An  active  dried  preparation  of  the  enzyme,  known  as  "  Arlco-urease," 
can  be  obtained  from  the  Arlington  Chemical  Company,  Yonkers,  N.Y., 
U.S.A.,  but  the  home-made  extract  is  very  much  less  costly  and  is  equally 
efficient. 

The  first  batch  of  enzyme  made  from  a  sample  of  the  Soya  bean  meal 
should  be  tested  by  making  a  2  per  cent,  solution  of  pure,  dry  urea,  and 
estimating  the  amount  in  0-5  cc.  of  this  by  the  method  described  below.  The 
result  obtained  should  be  within  4  per  cent,  of  theory,  the  slight  deficiency 
being  generally  due  to  impurities  in  the  urea. 

Method.  See  that  the  tube  B  (fig.  44)  and  the  narrow  tube  that 
goes  into  it  have  been  well  washed,  and  are  quite  free  from  any  of  the 
alkaline  carbonate  used  in  a  previous  experiment. 

(i.)  Measure  0-5  cc.  of  the  urine  into  B,  using  an  accurate 
Ostwald  pipette  (fig.  48).  If  the  urine  is  known  to  be  a  very  dilute 
one,  i  or  even  2  cc.  can  be  taken. 

(ii.)  Add  2  cc.  of  the  extract  of  Soya  bean  and  3  cc.  of  water, 
washing  the  traces  of  urine  down  to  the  bottom  of  the  tube  with  these 
two  fluids.  Lightly  shake  to  mix. 

(iii.)  Add  2  drops  of  caprylic  alcohol,  to  prevent  subsequent 
foaming. 

(iv.)     Fit  the  rubber  stopper  with  the  tubes  it  carries. 


*  Soya  bean  meal  can  be  obtained  from  Messrs.  Baird  and  Tatlock, 
London. 


336  ANALYSIS    OF   URINE.  [CH.  XIII. 

(v.)  Into  E  (or  the  flask  shewn  in  fig.  45)  measure  20  cc.  of  the 
standard  sulphuric  acid,  fit  the  stopper  and  connect  up  E  to  D. 

(vi.)  Immerse  the  tube  B  in  a  beaker  or  can  of  water  at  a 
temperature  of  about  45°  C.,  and  leave  it  undisturbed  for  10  to  12 
minutes. 

(vii.)  Remove  the  tube  from  the  bath,  connect  up  E  to  the 
pump,  and  B  to  the  wash  bottle  A,  and  send  a  strong  air  current 
through  the  apparatus  for  i  minute  to  sweep  over  any  ammonia 
that  may  have  escaped  from  the  fluid  and  be  present  in  the  air 
of  B. 

(viii.)  Stop  the  air  current  and  disconnect  the  entry  and  exit 
tubes  of  B.  Remove  the  stopper  with  these  tubes  and  place  it  on 
the  bench  in  such  a  way  that  the  small  amount  of  fluid  on  the  end 
of  the  entry  tube  is  not  lost. 

(ix.)  Add  4  to  5  grams,  of  pure  dry  potassium  carbonate  to  B, 
roughly  measuring  it  with  a  suitable  spoon,  immediately  replace  the 
stopper  and  connect  up  the  apparatus.  Send  a  slow  air  current 
through  for  2  minutes  and  then  a  rapid  current  for  12  minutes. 

(x.)     Proceed  as  described  in  Ex.  399  (iv.), 

Calculation.  Determine  the  percentage  of  nitrogen  in  the  form  of 
ammonia  and  urea  as  described  for  Kjeldahl's  method,  p.  323.  The  result 
thus  obtained  is  the  mgms.  of  (urea  +  ammonia)  N.  in  0-5  cc.  urine  =  Ua. 

So  mgms.  of  (urea  +  ammonia)  N.  in  100  cc.  =  200  x  Ua. 

From  Ex.  399,  the  mgms.  of  ammonia  N.  in  100  cc.  =  20  A. 

So  mgms.  of  urea  N.  in  TOO  cc.  =  (200  x  Ua)  -  20  A  =  U. 

Since  60  grams,  of  urea  contain  28  grams,  of  nitrogen,  the  grams,  of  urea  in 

60          i 
joo   cc.   urine  =  U  x        x  ^^  =  U  x  0-00214    (log.   3*3310)- 


E.     Creatinine  and  Creatine. 

The  methods  that  are  universally  adopted  are  based  on  Jaffe's  test  for 
creatinine  (Ex.  227)  as  applied  by  Folin  for  colorimetric  estimation.  Creatine 
does  not  reduce  picric  acid,  but"  is  converted  to  creatinine  either  by  heating 
with  hydrochloric  or  picric  acid.  The  combined  (creatinine  +  creatine)  is 
then  estimated  colorimetrically.  Folin  and  Doisy*  have  recently  pointed  out 
that  considerable  errors  may  occur  if  impure  picric  acid  is  used,  especially  in 


*  Folin  and  Doisy,  Journ.  Biol.  Chem.,  xxviii.,  p.  349. 


CH.  XIII.]  CREATININE.  337 

the  case  of  the  estimation  of  creatine.  They  give  a  method  of  purifying  the 
very  doubtful  specimens  of  wet  picric  acid  that  are  at  present  on  the  market. 
A  much  less  troublesome  method  is  given  on  p.  251,  but  for  observations  on  the 
excretion  of  creatine  in  pathological  conditions  it  would  be  safer  to  follow  Folin< 

Graham  and  Poulton*  have  shewn  that  the  presence  of  aceto-acetic  acid 
in  the  urine  inhibits  the  reaction  with  creatinine,  so  that  the  estimations  are 
too  low.  This  acid  is  destroyed  by  the  heating  required  for  the  estimation  of 
(creatinine  +  creatine),  so  that  the  result  of  the  analysis  always  makes  it 
appear  as  if  creatine  were  present.  They  give  a  method  for  the  removal  of 
aceto-acetic  acid,  which  must  be  followed  when  the  urine  gives  a  distinct 
Rothera's  test.  They  are  unable  to  confirm  the  statement  that  creatine  is 
found  in  the  urine  as  a  result  of  carbohydrate  starvation.  Tt  only  appears 
to  be  present  if  faulty  analytical  procedures  are  adopted,  aceto-acetic  acid 
always  being  found  in  carbohydrate  starvation. 

402.    The  estimation  of  creatinine  (Folin).f 

Principle.  A  measured  amount  of  the  urine  is  treated  with  picric  acid 
and  caustic  soda.  The  picric  acid  is  reduced  to  picramic  acid  in  the  cold  by 
the  creatinine  present,  glucose  having  no  effect  in  the  cold  (see  Exs.  108  and 
227).  A  known  amount  of  creatinine  is  similarly  treated  and  the  solutions 
compared  in  a  colorimeter. 

Solutions  and  apparatus  required. 

1.  A  colorimeter,   see  p.   384. 

2.  Standard  solution  of  creatinine  zinc  chloride,     i -6106  gram,  of  the 
pure  recrystallised  zinc  compound  (see  p.  300)  is  dissolved  in  about  500  cc.  of 
distilled  water  and  100  cc.  of  Normal  hydrochloric  acid  and  the  volume  made 
up  to  i  litre  with  distilled  water,     i  cc.  contains  i  mgm.  of  creatinine.     The 
solution  is  quite  stable. 

3.  A  saturated  aqueous  solution  of  pure  picric  acid  (about  1-2  per  cent.) 
and  a  20  cc.  pipette  for  measuring  it. 

4.  10  per  cent,  caustic  soda,  which  can  be  measured  by  a  pipette  or 
burette. 

5.  Ostwald  pipettes  (fig.  48)  of  i  cc. 

6.  Two  100  cc.  measuring  flasks. 

Method.  Into  a  100  cc.  measuring  flask  (labelled  "  U ") 
measure  i  cc.  of  the  urine  by  means  of  an  Ostwald  pipette.  Add 
20  cc.  of  the  picric  acid  and  then  1-5  cc.  of  the  soda.  Allow  the 
mixture  to  stand  for  10  minutes  with  gentle  agitation.  As  soon  as 
the  mixture  has  been  made  measure  i  cc.  of  the  standard  creatinine 
solution  into  the  other  100  cc.  flask  (labelled  "  S  "),  add  the  picric 

*  Graham  and  Poulton,  Proc.  Roy.  Soc.,  Ixxxvii.,  B.,  p.  205. 
f  Journ.  Biol.  Chern.,  xvii.,  p.  470. 


338  ANALYSIS   OF    URINE.  [CH.  XIII. 

acid  and  soda  as  before  and  mix,  noting  the  time.  After  the 
flasks  have  each  stood  for  10  minutes  they  are  separately  filled  to 
the  mark  with  distilled  water  and  the  contents  well  mixed.  The 
solutions  are  then  compared  in  a  colorimeter  (see  p.  384),  the 
standard  being  set  at  15  mm. 

Should  "  U  "  read  below  10  mm.,  the  determination  must  be 
repeated,  using  i  cc.  of  an  accurately  diluted  urine,  say  I  in  2  or 
i  in  3.  Should  "  U  "  read  above  22  mm.  the  determination  must 
be  repeated  with  2  cc.  or  more  of  the  urine.  In  such  cases  there 
is  no  necessity  to  make  another  standard,  the  colour  being 
quite  permanent  for  hours. 

Calculation. 

Mg.  in  i  cc.  urine  Reading  of  "  S  "  15 

Mg.  in  i  cc.  standard  "~    Reading  of  "  U  "          Reading  of  "  U  " 

MS- in  '  cc'  =  Reading'of  "U"  =  Cn" 

If  more  or  less  than  i  cc.  of  urine  have  been  taken,  this  must  be  divided 
by  the  volume  of  urine  used. 

Grams,  of  creatinine  in  100  cc.  =  Cn  x  o-i. 

Since  129  grams,  of  creatinine  contain  42  grams,  of  nitrogen,  grams,  of 

creatinine-  N   in    100    cc.  =  Cn  x  o-i  x  - —  -  Cn  x  -0325    (log.    2-5126). 

403.    The  estimation  of  creatine  and  creatinine  (Folin). 

(i.)     Weigh  a  200  cc.  Erlenmeyer  flask  of  "  Duro  "  glass. 

(ii.)  Into  it  measure  the  amount  of  urine  that  contains  between 
0-7  and  1-5  mgm.  of  creatinine,  as  determined  by  the  previous 
exercise. 

(iii.)  Add  20  cc.  of  saturated  picric  acid  and  about  130  cc.  of 
water  and  a  few  pieces  of  broken  porcelain. 

(iv.)     Boil  gently  over  a  micro-burner  for  i  hour. 

(v.)     Increase  the  heat  and  boil  down  to  rather  less  than  20  cc. 

(vi.)  Weigh  the  flask  and  add  water,  if  necessary,  to  make  the 
total  weight  of  the  contents  equal  to  20-25  grams. 

(vii.)    Cool  in  running  water. 


CH.  XIII.]  CREATINE.  339 

(viii.)     Add  1-5  cc.  of  10  per  cent,  soda  from  a  burette  and 
allow  the  mixture  to  stand  for  10  minutes  with  gentle  agitation. 

(ix.)     Transfer  to  a  100  cc.  volumetric  flask  and  wash  out  with 
distilled  water  to  make  100  cc. 

(x.)     Estimate  colorimetrically  as  in  the  previous  exercise. 

Calculation.  This  is  the  same  as  in  the  previous  exercise,  proper  allow- 
ance being  made  for  the  volume  of  urine  used.  The  difference  between  the 
two  results  is  the  creatine,  which  is  usually  expressed  in  terms  of  creatinine. 
To  convert  this  to  creatine  it  should  be  multiplied  by 

=1-14  (log.  -0567). 


404.    The  estimation  of  creatine  and  creatinine  (Benedict).* 

Principle.  The  dehydration  of  creatine  to  creatinine  is  very  rapidly 
effected  by  evaporation  to  dryness  with  hydrochloric  acid.  A  little  lead  is 
added  to  inhibit  pigment  formation,  the  traces  of  hydrogen  evolved  preventing 
oxidation.  It  is  not  applicable  to  urines  containing  glucose. 

Method.  Into  a  small  beaker  measure  that  volume  of  urine 
that  contains  7  to  10  mgm.  of  creatinine.  Add  10  to  20  cc.  of  N. 
hydrochloric  acid  and  a  pinch  or  two  of  powdered  or  granulated  lead. 
Boil  down  over  a  small  free  flame  till  nearly  dry  and  then  evaporate 
to  complete  dryness  on  a  boiling  water  bath.  Add  10  cc.  of  hot  dis- 
tilled water  and  filter  through  a  small  plug  of  cotton  wool  into  a 
narrow  25  cc.  measuring  cylinder.  Wash  out  quantitatively  with 
two  successive  portions  of  about  4  cc.  of  hot  water.  Cool  by 
immersion  in  cold  water  and  make  the  volume  up  to  20  cc.  Measure 
2  cc.  into  a  100  cc.  volumetric  flask,  using  an  Ostwald  pipette.  Add 
20  cc.  of  saturated  picric  acid  and  1-5  cc.  of  a  10  per  cent,  solution 
of  soda  that  contains  5  per  cent,  of  Rochelle  salt  (to  prevent  the 
formation  of  a  cloud  due  to  traces  of  dissolved  lead)  .  After  standing 
for  10  minutes  with  gentle  agitation,  make  up  to  the  mark  with 
distilled  water.  A  standard  is  simultaneously  prepared  from  I  cc. 
of  the  standard  creatinine  solution,  20  cc.  of  picric  acid  and  1-5  cc. 
of  the  10  per  cent,  soda  containing  5  per  cent,  of  Rochelle  salt, 
diluted  to  100  cc.  after  standing  for  10  minutes.  The  two  solutions 
are  read  as  described  in  Ex.  402. 

*  Journ.  Biol.  Chern.,  xviii.,  p.  191. 


340  ANALYSIS   OF   URINE.  [CH.  XIII. 

Calculation.     If  (a)  cc.  of  urine  have  been  taken  originally,  the  amount 

actually  used  corresponds  to  ift.    If  the  standard  is  set  at  15  mm.,  and  the 

10 
"  U  "  tube  reads  at  U,  then  mg.  of  (creatine  +  creatinine)  in 

15  x  10 

i  cc.  -  r<u"  x  (fl). 

The  rest  of  the  calculation  is  the  same  as  that  of  the  previous  exercise. 

NOTE. — The  above  method  is  a  slight  modification  of  that  published  by 
Benedict,  but  it  does  not  differ  in  any  essential. 

405.    The  removal  of  aceto-acetic  acid  (Graham  and  Poulton). 

Principle.  Aceto-acetic  acid  is  converted  by  heat  to  acetone,  which  is 
distilled  off  at  low  pressure.  The  following  account  is  slightly  modified  from 
the  original. 

Solutions  and  apparatus  required. 
(i.)     A  10  per  cent,  solution  of  phosphoric  acid. 

(ii.)  A  solution  of  soda  of  such  a  strength  that  i  cc.  of  it  neutralises  i  cc. 
of  the  phosphoric  acid,  phenol  phthalein  being  used  as  the  indicator. 
A  15  per  cent,  solution  of  soda  is  a  convenient  starting  point  for  the 
preparation  of  this.  When  it  is  correctly  adjusted,  1-5  cc.  of  it  will 
neutralise  all  three  valencies  of  i  cc.  of  the  phosphoric  acid,  only  two 
of  which  are  neutralised  to  phenol  phthalein. 

(iii.)     A  suction  pump  and  gauge  (see  fig.  9,  p.  74). 

(iv.)  A  thick  walled  tube  (25  to  30  mm.  by  200  mm.)  similar  to  the  tube  E 
of  fig.  44,  but  in  place  of  F  is  substituted  a  tube  drawn  out  to  a  fine 
capillary,  which  must  reach  nearly  to  the  bottom  of  E.  (The 
upper  end  of  the  tube  may  be  fitted  with  a  piece  of  pressure  tubing 
and  a  screw  clip  similar  to  that  shown  in  C  of  fig.  8.) 

Method.  Into  the  tube  measure  10  cc.  of  the  urine  and  add  i  cc. 
of  the  10  per  cent,  phosphoric  acid.  Fit  the  stopper  carrying  the 
capillary  tube  and  connect  the  other  outlet  tube  to  the  tube  E  of 
the  apparatus  shewn  in  fig.  9  and  have  C  turned  to  make  connexion 
with  A.  Turn  on  the  pump  and  note  the  pressure  obtained,  which 
depends  on  the  size  of  the  capillary,  on  the  size  of  A  and  on  the 
water  pressure.  It  is  necessary  to  maintain  a  pressure  of  about 
210  mm.  of  mercury.  Immerse  the  tube  containing  the  acidified 
urine  in  a  water  bath  kept  between  65°  and  70°  C.,  and  leave  it  for 
about  three-quarters  of  an  hour,  seeing  that  the  temperature  does 
not  rise  above  70°  C.,  nor  the  pressure  fall  below  210  mm.  of  mercury. 
Release  the  pressure  by  turning  the  tap  C  to  connect  with  B,  and 
then  turn  off  the  water.  Disconnect  and  cool  the  solution  under  the 
tap.  Add  i  -5  cc.  of  the  standardised  soda  to  completely  neutralise 


CH.  XIII .1  URIC   ACID.  341 

the  phosphoric  acid  and  then  transfer  to  a  narrow  25  cc.  cylinder. 
Wash  out  with  water  to  make  a  total  volume  of  20  cc.  Mix  well  and 
estimate  the  creatinine  by  the  method  given  in  Ex.  402.  2  cc.  of 
the  solution  correspond  to  i  cc.  of  the  urine. 

F.     Uric  Acid. 

Most  of  the  methods  employed  at  present  are  based  on  one  of  two  main 
principles.  The  first  is  on  Hopkins'  ammonium  chloride  method  ;  the  other 
is  colorimetric  with  Folin's  reagent.  The  author's  experience  with  all  modifi- 
cations of  the  latter  has  been  so  unfavourable  that  it  has  been  reluctantly 
abandoned.  It  is  possible  that  the  difficulty  of  obtaining  reliable  chemicals 
accounts  for  many  of  the  troubles,  but  the  greatest  care  in  this  respect  has  not 
been  rewarded  with  success. 

Hopkins'  is  the  standard  method.  It  requires  skill  and  practice  to  get 
good  results,  but  it  is  absolutely  reliable.  The  modification  of  it  introduced 
by  Folin  and  Schaffer  is  a  concession  to  the  unskilful  manipulator,  but  it  has 
the  disadvantage  of  an  allowance  of  3  mgms.  for  the  ammonium  urate  not 
precipitated  by  ammonium  sulphate.  The  author  humbly  suggests  that  this 
is  an  averaging  of  results,  for  comparisons  with  Hopkins'  original  method  and 
also  with  the  modification  described  below,  seem  to  indicate  that  with  certain 
specimens  of  urine  the  allowance  should  be  smaller  or  greater  than  this.  It  is 
always  the  same  for  a  given  specimen  of  urine,  suggesting  that  some  unknown 
factor  affects  the  solubility  of  ammonium  urate  under  the  conditions  of  the 
experiment. 

It  is  an  objection  to  Hopkins'  method  that  the  result  cannot  be  obtained 
rapidly,  as  the  solution  must  be  allowed  to  stand  over-night  for  the  whole  of 
the  uric  acid  to  crystallise  out.  This  inconvenience  is  also  a  feature  of  the 
Folin-Schaffer  modification.  Many  attempts  have  been  made  to  titrate  the 
original  precipitate  of  ammonium  urate,  but  they  have  not  been  very  suc- 
cessful owing  to  the  difficulty  of  removing  the  chlorides  which  also  titrate  with 
the  permanganate  in  acid  solution.  It  is  generally  stated  that  the  addition  of 
manganese  sulphate  prevents  the  action  of  the  chlorides.  By  accidentally 
using  magnesium  sulphate  on  one  occasion  the  author  was  led  to  investigate 
carefully  the  extent  to  which  the  presence  of  chlorides  interfere  with  a  correct 
result.  It  was  found  that  moderate  concentrations  have  no  effect,  owing  to 
the  great  rate  at  which  uric  acid  is  oxidised  compared  to  the  low  velocity 
of  the  reaction  between  chlorides  and  permanganate.  As  a  result  of  a  con- 
siderable amount  of  work  the  modification  described  below  was  elaborated. 
The  final  result  can  be  obtained  in  i£  hours,  and  in  the  hands  of  the  author 
agrees  to  i  mgm.  per  100  cc.  with  that  of  Hopkins'  original  method.  It  has 
been  regularly  used  in  class  work  for  the  past  4  years,  and  presents  little 
difficulty  to  the  average  student.  But  it  must  be  admitted  that  it  has  not 
been  tested  with  a  large  number  of  pathological  urines,  and  for  that  reason  it  is 
possible  that  in  certain  cases  it  will  only  yield  approximate  results.  There  is 
no  a  priori  reason  why  it  should  fail  more  than  other  methods. 

406.    Uric  acid  (Cole's  modification  of  Hopkins'  method). 

Principle.  The  urine  is  treated  with  colloidal  iron  to  remove  an  unknown 
substance  that  is  precipitated  by  ammonium  chloride.  The  filtrate  is  treated 
with  solid  ammonium  chloride  and  made  strongly  alkaline  with  ammonia. 


342  ANALYSIS   OF    URINE.  fCH.  XIII. 

The  uric  acid  is  rapidly  and  quantitatively  precipitated  as  ammonium  urate. 
This  is  filtered  off,  washed  with  ammonium  sulphate  to  remove  the  greater 
part  of  the  chlorides,  dissolved  in  hot  sulphuric  acid  and  titrated  with  standard 
potassium  permanganate.  The  end  point  is  reached  when  a  momentary  pink 
flush  is  seen  over  the  whole  body  of  the  fluid.  This  marks  a  stage  when  the 
rate  of  oxidation  of  the  uric  acid  suddenly  decreases.  Up  to  this  point  i  cc. 
of  0-05  N.  permanganate  is  found  empirically  to  correspond  to  3-7  mg.  of  uric 
acid.  The  chemical  changes  involved  in  the  oxidation  have  not  yet  been 
determined. 

Solutions  and  reagents  required. 
(i.)     Colloidal  (dialysed)  iron,  0-6  per  cent, 
(ii.)     Pure,  dry,  recrystallised  ammonium  chloride. 

(lii.)  Washing  fluid.  100  grams,  of  pure  ammonium  sulphate  are  dissolved 
in  about  800  cc.  of  distilled  water,  10  cc.  of  strong  ammonia  are  added 
and  the  volume  made  up  to  i  litre  with  water.  It  is  convenient 
to  use  this  from  a  wash  bottle  with  a  fine  jet. 

(iv.)  45  per  cent,  sulphuric  acid  (by  volume) .  To  500  cc.  of  distilled  water  in 
a  large  flask  cautiously  add  450  cc.  of  pure  concentrated  sulphuric 
acid,  cooling  at  intervals.  Cool  thoroughly  under  the  tap  and 
make  up  the  volume  to  1,000  cc. 

(v.)  0-05  N.  potassium  permanganate.  Dissolve  1-58  gram,  of  the  pure 
salt  in  distilled  water  and  make  the  volume  up  to  1,000  cc.  Care 
must  be  taken  to  see  that  the  whole  of  the  solid  has  dissolved  before 
the  solution  is  used.  It  can  be  titrated  against  pure  ammonium 
oxalate  as  described  on  p.  132.  If  Ox.  be  the  weight  of  the  oxalate 
taken  in  grams,  (about  0-15)  and  P  the  volume  of  permanganate 

required,   then  the  normality  is   p  x  O-O7IOS  =  Pn' 

i  cc.  of  0-05  N.  permanganate  "3-7  mgms.  uric  acid. 

_  3-7  x  Pn 
i  cc.  of  PnN.  permanganate  —  — ^7— —  mgms. 

Method.  Measure  1 50  cc.  of  the  urine  into  a  200  cc.  beaker, 
marking  the  100  cc.  level  by  means  of  a  label.  Add  30  cc.  of  the 
colloidal  iron,  stirring  well  during  the  addition.  Filter  through  two 
dry  papers  into  two  dry  flasks  (two  being  used  to  save  time) .  When 
at  least  100  cc.  of  the  nitrate  has  been  collected,  carefully  measure 
the  amount  taken  and  transfer  it  to  the  marked  beaker,  which  has 
been  previously  washed  and  drained.  It  is  convenient  to  take  100  cc. 
but  with  dilute  urines  it  is  better  to  take  120  or  150  cc.  For  every 
10  cc,  of  the  filtrate  taken  weigh  out  2  grams,  of  the  solid  ammonium 
chloride  ;  add  this  to  the  beaker  and  stir  well.  When  it  has  dis- 
solved add  3  cc.  of  concentrated  ammonia  and  stir  again.  Stir  at 
intervals  for  20  minutes,  then  remove  the  rod  and  let  it  rest  on  the 
rim  of  the  beaker.  When  the  bulk  of  the  precipitate  has  settled 


CH.  XIII.] 


URIC   ACID. 


343 


the  urate  is  filtered  off  either  through  a  plain  paper,  or  more  rapidly 
by  moderate  suction  through  a  paper  and  paper  pulp  supported  on  a 
perforated  plate  (20  to  25  mm.  diameter)  resting  in  a  funnel,  as 
shewn  in  the  accompanying  figure.  The  filter  is  prepared  by  cutting 
a  piece  of  filter  paper  rather  larger  than  the  disc,  placing  this  in 
position,  moistening  it  and  applying  suction  by  a  pump.     Any 
creases  round  the  edge  are  flattened  out  by  the  point  of  a  pencil. 
The  pressure  being  released 
a  little  paper  pulp  is  poured 
on  to  the  disc  and  allowed 
to  settle   and   then   gentle 
suction  applied.     The  pulp 
will     completely    seal    the 
cracks  between  the  disc  and 
the  funnel.     It  is  advisable 
to    cut    another    circle    of 
paper,     smaller    than    the 
first,  and  to  place  it  on  the 
centre     of    the    pulp.      It 
prevents   the    latter   being 
washed  away  during  filtra- 
tion.  Filter  the  supernatant 
fluid  first,  taking  care  not 
to     disturb     the    bulk    of 
the    precipitate.       Do    not 
use  too  high  a  pressure,  as  this  drives  the  amorphous  ammonium 
urate  into  a  cake  which  renders  the  subsequent  washing  very  slow. 
The  pressure  can  be  regulated  by  use  of  the  apparatus  shewn  on 
page  74.      If  this  is  not  available,  a  T-piece  can  be  used,  as  shewn 
in  fig.  45a.     One  limb  of  this  is  connected  to  the  pump  and  the  other 
is  fitted  with  a  piece  of  pressure  tubing  and  a  spring  clip,  by  means 
of  which  the  pressure  can  be  instantaneously  released.     When  the 
main  mass  of  the  fluid  has  passed  through,  transfer  the  bulk  of  the 
precipitate,  but  do  not  suck  quite  dry.     Wash  out  the  precipitate 
remaining  in  the  beaker  with  the  ammonium  sulphate  solution  on  to 
the  filter  and  start  suction  again.     Do  this  twice  more,  finally 
sucking  the  precipitate  dry.     The  object  of  the  washing  is  to  remove 
as  much  ammonium  chloride  as  possible  from  the  precipitate,  paper 


Fig.  45A. 


344  ANALYSIS   OF    URINE.  [CH.  xill  . 

and  beaker.  Now  transfer  the  paper,  precipitate  and  disc  to  the 
marked  beaker,  by  means  of  a  glass  rod  which  has  a  fine  curved  point. 
Remove  the  funnel  from  the  filtering  flask  and  wash  it  down  into  the 
beaker  with  a  jet  of  hot  water.  Also  wash  the  pointed  glass  rod. 
Add  hot  water  to  make  a  total  volume  of  100  cc.,  as  indicated  by 
the  label.  Add  20  cc.  of  the  45  per  cent,  sulphuric  acid  and  stir 
with  a  thermometer.  The  whole  of  the  precipitate  must  be  dis- 
solved, if  necessary  by  the  aid  of  heat,  before  the  titration  is  com- 
menced. Cool  or  heat  to  65°  C.  Titrate  with  the  standard  perman- 
ganate, reading  the  meniscus  by  the  aid  of  a  lighted  match  held 
behind  the  burette.  The  permanganate  must  be  added  rather 
slowly,  with  constant  stirring.  The  end  point  is  reached  when  the 
addition  of  a  couple  of  drops  causes  a  faint  pink  flush  through  the 
whole  body  of  the  fluid.  A  very  considerable  addition  has  to  be 
made  for  the  pink  to  be  permanent,  but  the  empirical  valuation  of 
the  permanganate  is  based  on  the  end  point  described  above. 

Calculation.  Since  150  cc.  of  the  urine  were  treated  with  30  cc.  (one- 
fifth  volume)  of  colloidal  iron,  6  cc.  of  the  filtrate  from  this  correspond  to  5  cc. 
of  urine. 

If  u  cc.  of  the  filtrate  have  been  taken  and  .p  cc.  of  0-05  N.  permanganate 
used,  then  u  cc.  contain/)  x  3-7  mgms.  uric  acid,  and  100  cc.  of  urine  contain 

6      100          i  p 

p  x  3-7  x  jx  -jj-  x-^    gram.  =  -  x  0-444  gram. 


NOTE.  —  Whilst  this  edition  was  passing  through  the  press  the  author 
has  found  that  the  best  method  of  filtering  off  the  urate  is  to  use  a  Hirsch 
funnel  with  a  fixed  perforated  plate,  and  to  use  asbestos  instead  of  paper 
pulp.  The  filtering  mat  is  prepared  in  the  way  described  for  a  Gooch 
crucible  (seep.  389),  the  drying  being  omitted.  The  mat  with  the  layer  of 
urates  can  be  very  neatly  removed  and  the  whole  process  is  much  facilitated. 
The  time  required  for  the  estimation  is  considerably  overstated  above.  The 
whole  process  can  be  accomplished  in  40  minutes,  it  being  unnecessary  to 
allow  the  fluid  to  stand  for  more  than  10  minutes  after  the  addition  of  the 
ammonia. 


G.    Glucose. 

The  estimation  of  sugar  in  urine  when  there  is  a  considerable  amount 
present  can  be  carried  out  by  any  of  the  standard  methods,  as,  owing  to  the 
dilution  necessary  or  the  small  amount  required,  there  is  little  interference 
by  the  normal  urinary  constituents.  If  the  concentration  is  less  than  0-8  per 
cent,  the  author  prefers  to  use  the  Wood-Ost  method  (p.  131),  and  to  dilute 
the  urine  at  least  three  times  with  water.  Though  the  amount  of  permanga- 
nate required  is  small,  the  results  are  quite  satisfactory.  With  Benedict's 
method  of  direct  titration  (p.  127)  the  end  point  is  apt  to  be  very  uncertain 
with  low  concentrations  of  sugar.  The  polarimetric  method  referred  to  on 


CH.  xiir .]  GLUCOSE.  345 

page  127  is  of  great  service  when  a  large  number  of  diabetic  urines  have  to  be 
examined.  The  results  may  be  rather  low  owing  to  the  presence  of  the  laevo- 
rotatory  /3-oxy-butyric  acid. 

The  method  described  below  is  the  only  one  at  present  available  for  the 
estimation  of  such  small  amounts  of  glucose  as  are  present  in  normal  urine. 
It  is  included  in  the  hope  that  the  study  of  slight  variations  from  the  normal 
will  extend  our  knowledge  of  the  pathology  of  diabetes,  and  also  as  a  practical 
method  for  the  detection  of  lowered  tolerance  to  carbohydrates. 

407.  The  estimation  of  glucose  in  normal  urine  (Benedict  and 
Osterberg).* 

Principle.  The  urine  is  treated  with  mercuric  nitrate  and  neutralised 
with  sodium  bicarbonate.  The  creatinine,  urates,  etc.,  are  thus  removed. 
The  mercury  is  removed  by  means  of  zinc  and  the  sugar  estimated  by  the 
colorimetric  method  with  picric  acid. 

Solutions  and  apparatus  required.^ 

1.  Picric-pier  ate  mixture,  see  p.  251. 

2.  Standard  solution  of  glucose.     The  stock  solution  is  described  on 
p.  251.     5  cc.  of  this  are  diluted  to  make  50  cc.  with  distilled  water,     i  cc. 
contains  i  mg.  glucose. 

An  alternative  standard  can  be  prepared  from  pure  picramic  acid.  The 
stock  solution  is  described  on  p.  251.  105  cc.  of  this  are  treated  with  0-5  cc. 
of  20  per  cent,  sodium  carbonate  and  15  cc.  of  the  picric-picrate  mixture  and 
diluted  to  make  300  cc.  with  distilled  water.  The  colour  obtained  corresponds 
to  that  of  i  mg.  glucose  in  4  cc.  of  water,  treated  as  described  for  the  final  urine 
filtrates  and  the  coloured  solution  diluted  to  25  cc.  It  is  the  most  convenient 
standard  to  use,  as  time  is  saved  and  a  possible  error  of  measurement  avoided. 

3.  Sodium  carbonate  solution,  20  per  cent.,  see  p.  252. 

4.  Test-tubes  graduated  at  12-5  and  25  cc.,  see  p.  252. 

5.  Ostwald  pipettes,  see  p.  381. 

6.  A  colorimeter,  see  p.  384. 

7.  Mercuric  nitrate  solution,  see  A  solution,  p.  391.     On  no  account  must 
the  B  solution  be  used  as  a  substitute. 

Method.  Into  a  50  cc.  beaker  measure  20  cc.  of  the  urine  and 
then  20  cc.  of  the  mercuric  nitrate  solution.  Mix  and  add  solid 
sodium  bicarbonate  with  gentle  shaking.  Considerable  frothing 
occurs.  The  bicarbonate  can  be  added  fairly  freely  until  this 
ceases.  Stir  well  and  see  that  the  material  on  the  sides  of  the 
beaker  is  mixed  with  the  main  mass,  which  forms  a  kind  of  paste. 
Now  add  the  bicarbonate  until  the  fluid  reacts  just  alkaline  to 
litmus  paper.  Filter  at  once  through  a  dry  paper  into  a  small  dry 

*  Journ.  Biol.  Chem.,  xxxiv.,  p.  195. 

|  These  can  be  obtained  from  Messrs.  Baird  and  Tatlock,  London. 


346  ANALYSIS    OF   URINE.  [CH.  XIII. 

flask.  The  filtrate  should  be  quite  clear  and  colourless.  Add  a 
pinch  of  zinc  dust  and  2  drops  of  concentrated  hydrochloric  acid, 
shake  and  allow  it  to  stand  for  5  to  10  minutes.  Filter  through  a 
small  dry  filter  into  a  dry  test-tube. 

Measure  I  to  4  cc.  of  this  filtrate  (so  that  0-5  to  2  mg.  sugar  are 
taken)  into  one  of  the  graduated  tubes.  If  less  than  4  cc.  are  taken 
make  the  volume  up  to  exactly  4  cc.  with  distilled  water.  Add  I  cc. 
of  the  20  per  cent,  sodium  carbonate  and  4  cc.  of  the  picric-picrate 
mixture  and  plug  the  tube  with  cotton  wool.  If  the  standard  is  pre- 
pared from  glucose,  measure  I  cc.  (i.e.  i  mg.)  into  another  graduated 
tube  (marked  "  S  ").  Add  3  cc.  of  water,  i  cc.  of  the  sodium 
carbonate,  4  cc.  of  the  picric-picrate  mixture  and  plug  with  cotton 
wool.  Immerse  both  tubes  in  a  boiling  water  bath  and  note  the 
time.  After  exactly  10  minutes  remove  the  tubes  and  cool  thoroughly 
under  the  tap.  Dilute  the  "  S  "  tube  to  25  cc.  If  picramic  acid  is 
used  as  a  standard,  fill  a  tube  with  some  of  the  dilute  solution. 
Dilute  the  other  tube  to  12*5  cc.  or  to  25  cc.  depending  on  the  colour 
obtained.  If  on  diluting  to  25  c.c.  the  colour  is  still  much  darker 
than  the  standard,  the  experiment  must  be  repeated,  using  less  of 
the  final  filtrate  from  the  zinc  or  diluting  the  urine  and  starting  again 
from  the  beginning. 

When  the  two  colours  are  roughly^the  same,  compare  with 
the  standard  in  a  colorimeter  (see  p.  384),  setting  the  standard  at  a 
height  of  15  mm.  It  may  be  necessary  to  filter  the  solution  contain  - 
ing  the  urine  from  a  slight  precipitate  that  appears  on  heating  with 
the  alkali  and  picrate. 

Calculation.  This  depends  on  the  amount  of  final  filtrate  taken  and  on 
the  dilution.  Suppose  that  2  cc.  of  final  filtrate  were  taken  and  it  was  diluted 
to  1 2 -5  cc.,  and  that  the  reading  was  17-4  mm.,  against  the  standard  at  15. 
Now  2  cc.  of  the  filtrate  contain  i  cc.  of  urine,  and  the  standard  contains  i  mg. 
of  glucose  (or  corresponds  to  this  if  picramic  acid  be  used) .  Since  the  urinary 
solution  was  only  diluted  to  12-5  cc.,  whilst  the  standard  was  made  up  to 
25  cc.,  the  result  must  be  halved. 

mg.  of  glucose  in  i  cc.        15        i 
~T~  =  iT^T  x  2- 

In    general 

Reading  of  "  S  "       Volume  after  heating       ^ 
mg.  of  glucose  in  i  cc.  =  Readingof«Tj»  *  vtfS^litaSteli^d   x  25' 

15  12-5  2 

=  ^T4  *  -T  X  2-5' 


CH.  XIII.]  ACETONE   BODIES.  347 

NOTE.— The  above  method  gives  the  total  of  glucose  and  non-fermentable 
carbohydrate.  Benedict  and  Osterberg  give  the  following  for  the  determina- 
tion of  the  latter.  To  25  cc.  of  urine  (free  from  preservative)  in  a  cylinder  or 
test-tube  add  20  to  25  mg.  of  glucose  and  about  one-quarter  cake  of  yeast. 
Mix  well  and  allow  to  stand  in  the  incubator  at  35-38°  C.  for  1 8  to  20  hours. 
Decant  15  to  20  cc.  of  the  urine  and  determine  sugar  as  before  fermentation. 
The  difference  between  the  two  gives  the  fermentable  sugar. 


H.    The  Acetone  Bodies. 

A  very  large  number  of  methods  have  been  proposed,  and  in  this  case  the 
latest  is  undoubtedly  the  best,  since  Van  Slyke's  method  gives  the  #-oxy- 
butyric  acid  as  well  as  the  acetone  and  aceto-acetic  acid.  Since  the  j6-oxy- 
butyric  acid  usually  forms  about  75  per  cent,  of  the  total  acetone  bodies  ex- 
creted, its  estimation  is  of  the  utmost  importance  in  all  studies  related  to  the 
origin  and  excretion  of  these  substances.  The  Scott- Wilson  method  is  also 
described,  as  it  is  considerably  quicker  and  gives  a  good  indication  of  the 
condition  of  the  subject. 

408.     Total  acetone  bodies  (D.  Van  Slyke).* 

Principle.  The  urine  is  treated  with  copper  sulphate  and  lime  to  remove 
sugar  and  other  interfering  substances.  The  filtrate  is  boiled  with  mercuric 
sulphate  and  sulphuric  acid  under  an  inverted  condenser.  Potassium  dichro- 
mate  is  run  in  down  the  condenser  to  oxidise  the  oxy-butyric  acid  to  acetone, 
whilst  the  aceto-acetic  acid  is  very  rapidly  converted  to  acetone  by  the  influence 
of  the  hot  acid.  The  acetone  forms  an  insoluble  compound  with  mercury 
which  is  filtered  off  and  weighed  in  a  Gooch  crucible.  The  precipitate  can  be 
titrated  if  desired  by  a  method  described  in  the  original  paper. 

Solutions  required. 

1.  Copper  sulphate.     200  grams,  of  the  pure  crystalline  salt  are  dissolved 
in  water  and  made  up  to  i  litre. 

2.  Mercuric  sulphate  solution.     73  grams,  of  pure  red  mercuric  oxide  are 
dissolved  in  i  litre  of  4  N.  sulphuric  acid. 

3.  Sulphuric  acid.     To  500  cc.  of  distilled  water  in  a  large  flask  cautiously 
add  500  cc.  of  concentrated  sulphuric  acid.     Cool  thoroughly  under  the  tap 
and  make  up  to  i  litre  with  distilled  water.     Titrate  a  portion  of  2  cc.  with 
N.  soda  (or  titrate  5  cc.  with  5  N.  soda)  and  adjust  to  17  N.  if  necessary. 

4.  Calcium   hydroxide  suspension.     Mix  100   grams,   of    pure   "  light '' 
calcium  hydroxide  with  i  litre  of  distilled  water. 

5.  Potassium  dichromate.     Dissolve  50   grams,  in  water  and  make  up 
to  i   litre. 

Method,  (i.)  Measure  25  cc.  of  the  urine  into  a  250  cc. 
volumetric  flask.  Add  100  cc.  of  distilled  water,  50  cc.  of  the  copper 
sulphate  solution  and  mix.  Then  add  50  cc.  of  the  calcium  hydroxide 
suspension  (previously  well  shaken)  and  shake  well.  Test  the 

*  Journ.  Biol.  Chem.,  xxxii.,  p.  455. 


ANALYSIS   OF    URINE.  fCH.  XIII. 

reaction  with  litmus.  If  not  alkaline  add  more  of  the  calcium 
hydroxide.  Dilute  to  the  mark  and  allow  it  to  stand  for  at  least 
.30  minutes.  Filter  through  a  dry  folded  paper  into  a  dry  flask. 
This  will  remove  up  to  8  per  cent,  of  glucose.  If  more  than  this  is 
present,  the  urine  must  be  diluted  to  bring  it  down  to  8  per  cent. 
If  glucose  is  present  in  the  nitrate  a  yellow  (not  white)  precipitate 
will  appear  if  a  little  of  it  is  boiled  in  a  test-tube. 

(ii.)  Connect  up  a  500  cc.  Erlenmeyer  flask  with  a  straight 
reflux  condenser,  as  shewn  on  p.  72.  Into  the  flask  measure  25  cc. 
of  the  urine  filtrate,  100  cc.  of  water,  10  cc.  of  the  17  N.  sulphuric 
acid  and  35  cc.  of  the  mercuric  sulphate  solution.  Connect  up  to  the 
condenser  and  heat  to  boiling  over  a  free  flame  (not  over  a  sand 
bath) .  When  boiling  has  begun  add  5  cc.  of  the  dichromate  solution, 
running  this  down  the  condenser  tube.  Allow  the  mixture  to  boil 
gently  for  ij  hours. 

(iii.)  Cool  the  solution  and  filter  off  the  precipitate,  using  a 
weighed  Gooch  crucible  (see  notes  to  Ex.  410).  Wash  out  the  flask 
with  cold  water,  of  which  about  200  cc.  in  all  should  be  used.  Dry 
the  crucible  in  an  hot-air  oven  at  110°  C.  for  an  hour.  Allow  the 
crucible  to  cool  down  to  air  temperature  and  weigh  again. 

Calculation.  If  25  cc.  of  the  filtrate  (representing  2-5  cc.  of  the  urine)  are 
used,  i  gram,  of  precipitate  corresponds  to  2-48  grams,  of  total  acetone  bodies 
per  100  cc.  in  terms  of  acetone.  This  is  on  the  assumption  that  the  molecular 
proportion  of  the  acetone  bodies  in  the  form  of  j8-oxy-butyric  acid  is  75  per 
cent.,  the  usual  figure. 

409.  /3-oxy-butyric  acid.  The  acetone  and  aceto-acetic  acid 
are  first  boiled  off  and  then  the  estimation  conducted  as  before. 
Measure  25  cc.  of  the  filtrate  into  the  open  flask,  add  100  cc.  of 
water  and  2  cc.  of  the  sulphuric  acid.  Boil  gently  for  10  minutes 
with  a  free  flame.  Cool  and  transfer  to  a  measuring  cylinder  and 
note  the  volume.  Return  it  to  the  flask  and  add  water  to  the  cylinder 
to  make  a  total  volume  of  127  cc.  Add  8  cc.  of  the  sulphuric  acid 
and  35  cc.  of  the  mercuric  sulphate.  Connect  up  to  the  condenser 
and  boil.  When  boiling  add  5  cc.  of  the  dichromate  and  allow  the 
mixture  to  boil  gently  for  I J  hours.  Then  proceed  as  in  Ex.  408  (iii) . 

Calculation,  i  gram,  of  precipitate  corresponds  to  2-64  grams,  of  £-oxy- 
butyric  acid  per  100  cc.,  reckoned  as  acetone :  to  convert  to  /3-oxy-butyric 

104 
acid  multiply  by  —-  =  1-793. 


CH.  XIII.]  ACETONE  BODIES.  349 

410.  Acetone    and   aceto-acetic  acid.     This  can  be  found 
by  difference  between  the  two  previous  exercises,  or  it  can  be 
determined  separately  by  the  method  adopted  for  total  acetone 
bodies,  except  that  (i)  no  dichromate  is  added,  and  (2)  the  boiling  is 
continued  for  not  less  than  30  nor  more  than  45  minutes. 

Calculation,  i  gram,  of  precipitate  corresponds  to  2-0  grams,  of  acetone 
and  aceto-acetic  acid  per  cent.,  reckoned  as  acetone. 

NOTES. — i.  Van  Slyke  also  gives  a  method  of  titrating  the  mercury 
precipitate.  It  is  presumably  quicker,  since  it  is  not  necessary  to  prepare  and 
dry  the  Gooch  crucible.  The  author  has  no  experience  of  it. 

2.  The  Gooch  crucible  should  be  of  25  to  50  cc.  capacity,  and  fitted  as 
shewn  on  page  259.     The  asbestos  mat  can  be  prepared  as  described  on  page 
132,  except  that  it  should  be  thicker  than  stated  there.     It  is  thoroughly 
washed  and  firmly  sucked  down  and  dried  in  a  steam  oven  or  a  hot-air  oven 
at  110°  C.     It  is  allowed  to  cool  in  the  air  and  weighed.     It  is  then  fitted  to 
the  rubber  cup  and  some  distilled  water  carefully  added  and  sucked  gently 
through  before  the  mercury  precipitate  is  filtered  off.     Several  precipitates 
can  be  collected  and  weighed  one  after  another.     (See  also  p.  389.) 

3.  The  reagents  should  be  tested  by  performing  an  experiment  with 
distilled  water  instead  of  urine,  starting  with  the  copper  sulphate  treatment. 
No  precipitate  whatever  should  be  obtained.     Van  Slyke  gives  a  caution  that 
this  test  should  not  be  omitted. 

4.  A  blank  determination  of  precipitate  from  other  substances  in  urine 
ether  than  the  acetone  bodies  may  be  made  by  following  the  procedure  of 
Ex.  409,  except  that  5  cc.  of  water  are  substituted  for  the  dichromate  and 
the  boiling  period  under  the  condenser  is  strictly  limited  to  45  minutes.     The 
weight  of  precipitate  obtained  is  deducted  from  that  found  in  any  estimation 
of  the  acetone  bodies.     It  is  usually  so  small  that  it  can  be  neglected,  except 
in  cases  where  only  small  amounts  of  the  acetone  bodies  are  present. 

411.  The  estimation  of  acetone   and   aceto-acetic   acid  in 
urine  by  the  method  of  Scott- Wilson.* 

Principle.  The  urine  is  distilled  into  an  alkaline  solution  of  silver 
mercuric  cyanide.  The  aceto-acetic  acid  is  decomposed  into  acetone,  which 
passes  over  with  any  preformed  acetone  into  the  cyanide.  An  insoluble  keto- 
mercuric-cyanide  compound  is  formed.  This  is  filtered  off,  dissolved  in  acid, 
and  the  amount  of  mercury  determined  by  titration  with  standard  thiocyanate. 
From  the  amount  of  mercury  present  the  total  amount  of  acetone  in  the  urine 
taken  can  be  calculated. 

Solutions  required. 

i.  Silver  mercury  cyanide.  Dissolve  9  grams,  of  pure  caustic  soda  and 
0-5  gram,  of  mercuric  cyanide  in  60  cc.  of  distilled  water.  Add  20  cc.  of  0-7268 
per  cent,  silver  nitrate  slowly  with  constant  stirring.  If  necessary  filter 
through  a  layer  of  washed  asbestos  in  a  Gooch  crucible.  The  silver  nitrate 
solution  is  prepared  by  diluting  5  cc.  of  the  standard  silver  nitrate  used  in  the 
next  exercise  with  15  cc.  of  distilled  water. 

*  Journ.  of  Physiology,  xlii.,  p.  444. 


35°  ANALYSIS   OF   URINE.  [CH.  XIII. 

2.  Acid  mixture.    Strong  nitric  acid  40  cc. 

Strong  sulphuric  acid      5  cc. 
Distilled  water  55  cc. 

3.  O-2    N.  potassium  permanganate.     6-324   grams,    of   permanganate 
dissolved  in  water  and  the  volume  made  up  to  i  litre.     There  is  no  necessity 
to  standardise  it  exactly. 

4.  Standardised  solution  of  potassium  thiocyanate.     Dilute  125  cc.  of  the 
stock  solution  described  in  Ex.  412  to  make  i  litre  with  distilled  water. 
Standardise  the  stock  solution  against  standard  silver  nitrate  as  described 
in  Ex.  412.     Divide  21-4  by  the  number  of  cc.  of  KCNS  required  for  10  cc.  of 
silver  nitrate. 

The  result  =  mg.  of  Hg  per  cc.  of  the  diluted  KCNS. 

5.  A  saturated  aqueous  solution  of  iron  alum. 

Method. 

i.  The  distillation  of  the  acetone.  Use  the  apparatus  shewn 
in  fig.  46.  Into  flask  A  measure  an  amount  of  urine  that  yields 
between  0-4  and  2  mg.  acetone.  Add  water  to  make  the  volume 


Fig.  46.     Apparatus  for  the  estimation  of  acetone. 

A.  "  Duro  "  flask.  B.  Solid  glass  rod  for  sealing  tube.  C.  "  Duro  "  flask. 
D.  Glass  tube  connected  by  rubber  to  condenser  tube.  E.  Erlenmeyer  flask. 
F.  Liebig  condenser. 

up  to  about  100  cc.  and  then  i  cc.  of  strong  sulphuric  acid.  Into 
flask  C  place  10  cc.  of  40  per  cent,  caustic  soda  and  a  few  glass 
beads.  Into  E  place  20  cc.  or  more  of  the  silver  mercury  cyanide 
reagent  (there  must  be  at  least  25  cc.  to  each  mg.  of  acetone). 
Close  the  tube  with  the  glass  rod  B  and  then  light  the  burners. 
The  soda  in  C  must  boil  before  the  fluid  in  A.  The  soda  is  kept 


CH.  XIII.]  ACETONE   BODIES.  351 

just  boiling  whilst  A  is  allowed  to  boil  briskly.  The  first  appear- 
ance of  turbidity  in  E  is  noted  and  the  distillation  allowed  to 
proceed  for  another  six  minutes.  Remove  plug  B  and  turn  out 
the  flames.  Detach  tube  D  from  the  condenser  and  wash  it  with 
a  jet  of  distilled  water  into  E.  Allow  the  fluid  to  stand  for  10 
minutes. 

2.  Filtration  of  the  mercury  compound.     Use  an  apparatus 
similar  to  that  shewn  in  fig.  33,  p.  259.     The  Gooch  crucible  should 
be  of  10  cc.  and  the  filtering  flask  of  250  cc.  capacity.     First  prepare 
the  crucible.     Cut  a  filter  paper  slightly  larger  than  the  bottom  of 
the  crucible,  place  it  in  position  and  moisten  it.    Then  pour  in  a 
suspension  of  washed  asbestos  fibre  and  form  a  mat  of  this  by 
applying  suction.     A  small  amount  of  a  suspension  of  washed 
powdered  pumice  should  next  be  filtered  to  partly  close  the  pores 
of  the  filter. 

Filter  the  fluid  in  flask  E  through  this.  If  the  first  portions  of 
the  filtrate  are  cloudy  they  must  be  refiltered.  Wash  the  precipitate 
with  cold  water  till  free  from  silver. 

3.  Solution  of  the  mercury  compound.     By  means  of  a  glass  rod 
with  a  pointed  hook  transfer  the  filter  mat  to  an  Erlenmeyer  flask. 
Place  the  crucible  in  the  neck  of  the  flask  and  wash  it  through  into 
the  flask  with  10  cc.  of  the  acid  mixture.    The  point  of  the  rod  should 
also  be  washed  with  a  little  of  this  solution.     Add  i  cc.  of  the 
potassium  permanganate  and  boil  till  colourless.     Add  more  of  the 
permanganate,  a  few  drops  at  a  time,  till  the  brown  tinge  persists  in 
the  boiling  mixture  for  about  two  minutes.     Discharge  the  colour 
by  the  addition  of  a  few  drops  of  yellow  nitric  acid. 

4.  Titration  of  the  mercury.     Cool  thoroughly  under  the  tap 
and  add  2  cc.  of  the  iron  alum.     Titrate  with  the  diluted  thiocyanate 
against  a  white  ground  until  a  very  faint  pinkish  brown  tinge  is 
permanent.     The  end  point  is  quite  sharp,  but  it  must  be  noted  that 
after  it  is  reached  a  considerable  amount  of  thiocyanate  can  be 
added  without  appreciably  darkening  the  tint. 

Calculation.     From  the  amount  of  thiocyanate  required  the  amount  of 
mercury  can  be  found. 

i  mg.  Hg  =  0-0606  mg.  acetone. 


352  ANALYSIS   OF   URINE.  [CH.  XIII. 

Example.     10  cc.  standard  silver  =  20-2  cc.  of  stock  KCNS. 
So  i  cc.  dilute  KCNS  =  ^|  =  i  -06  mg.  Hg. 

2  cc.  of  diabetic  urine  taken.     Mercury  in  ppt.  required  24-2  cc.  KCNS. 

So  mercury  =  24-2  x  1-06  =  25-65  mg. 

So  total  acetone  in  2  cc.  =  25-65  x  0-0606  =  1-55  mg. 

So  total  acetone  in  100  cc.  =  1-55  x  50  =  77-5  mg. 

NOTES. — i .  The  original  description  gives  a  different  method  of  standard- 
ising the  thiocyanate  solution,  by  titration  against  a  solution  of  mercuric 
sulphate,  which  itself  is  standardised  gravimetrically. 

2.  The  total  amount  of  acetone  bodies,  reckoned  as  acetone,  can  usually 
be  arrived  at  by  multiplying  the  figure  found  by  the  above  analysis  by  4-5, 
but  this  can  only  be  regarded  as  a  rough  approximation. 

I.    Chlorides. 

The  usual  method  for  the  estimation  of  chlorides  in  urine  is  Volhard's, 
which  is  described  on  page  199  in  connexion  with  the  estimation  of  gastric 
juice.  The  method  given  below  is  precisely  similar.  The  silver  nitrate 
solution  employed  is  of  a  different  strength  and  the  thiocyanate  is  not  made 
up  to  any  defined  concentration,  but  standardised  against  the  silver.  If 
preferred  the  solutions  described  on  page  199  can  be  employed,  i  cc.  of  the 
o-i  N.  silver  nitrate  being  equivalent  to  0-00355  gram,  of  chlorine  and  0-00585 
gram,  of  sodium  chloride.  If  this  weaker  silver  solution  is  used,  25  or  30  cc. 
of  it  should  be  taken  for  10  cc.  of  urine. 

A  method  that  is  rapid,  convenient  and  accurate,  is  that  of  Larrson, 
but  it  is  now  difficult  to  obtain  satisfactory  charcoal.  Recently,  however, 
the  author  has  been  presented  with  a  specimen  of  charcoal  that  was  prepared 
for  use  in  gas  masks.  It  seems  to  be  an  extremely  good  absorbent,  superior 
to  the  best  German  products  and  admirably  adapted  for  all  kinds  of  analy- 
tical work.  The  method  is  described  in  the  hope  that  a  satisfactory  product 
will  soon  be  on  the  market.  In  that  case  Volhard's  method  would  be  super- 
seded by  Larrson's. 

412.    The  estimation  of  chlorides  by  Volhard's  method. 

Principle.     See  page  199. 

Reagents  required. 

1.  Standard  silver  nitrate  solution  prepared  by  dissolving  29-063  grams, 
of  pure  fused  silver  nitrate  in  distilled  water  and  filling  up  accurately  to  one 
litre.     The  solution  should  be  kept  in  the  dark. 

i  cc.  corresponds  to  -01  gram.  NaCl  (-00606  gram.  Cl). 

2.  Solution  of  potassium  thiocyanate  made  by  dissolving  8  grams,  of  the 
salt  in  a  litre  of  distilled  water. 

3.  Pure  nitric  acid,  quite  free  from  chlorine. 

4.  A  concentrated  solution  of  iron  alum. 


CH.  XIII.]  CHLORIDES.  353 

Standardisation  of  the  thiocyanate.  In  a  beaker  place  10  cc.  of  the 
silver  nitrate,  accurately  measured  :  add  5  cc.  of  pure  nitric  acid,  5  cc.  of  iron 
alum  and  80  cc.  of  distilled  water.  Titrate  the  whole  with  the  thiocyanate 
from  a  burette  until  a  faint  permanent  red  tinge  is  obtained.  Note  the 
amount  required  for  the  10  cc.  of  silver  nitrate. 

Method.  In  a  100  cc.  cylinder  or  measuring  flask  place  10  cc. 
of  urine,  accurately  measured  by  a  pipette,  20  cc.  of  the  standard 
silver  solution,  also  accurately  measured,  about  4  cc.  of  pure  nitric 
acid,  and  5  cc.  of  the  iron  alum.  Add  distilled  water  to  the  100  cc. 
mark,  and  mix  thoroughly  by  pouring  into  a  beaker  and  stirring  well. 
Filter  off  the  precipitated  silver  chloride  through  a  dry  paper  into  a 
dry  vessel.  Of  the  filtrate  take  50  cc.,  accurately  measured,  and 
titrate  it  with  the  potassium  thiocyanate  solution  till  a  faint  per- 
manent red  tinge  is  obtained. 

NOTES.— i.  It  is  very  important  to  remember  to  add  the  nitric  acid.  It 
renders  the  silver  chloride  insoluble  and  prevents  the  precipitation  of  the  silver 
compounds  of  the  purine  bases  in  those  cases  in  which  the  urine  is  alkaline. 

2.  Some  workers  titrate  without  filtering  off  the  silver  chloride,  but  the 
end  point  is  apt  to  be  uncertain  owing  to  the  decomposition  of  the  chloride 
by  the  thiocyanate. 

Calculation  and  Example. 

19-6  cc.  of  the  KCNS  were  required  for  10  cc.  of  the  AgNO3. 

10 
So  i  cc.  of  the  KCNS  ~  ^^5  =  0-51  cc.  AgNOa. 

50  cc.  urinary  filtrate  required  n-6  cc.  KCNS, 

So  100  cc.  urinary  filtrate  would  require  23*2  cc.  KCNS  and  would 
therefore  contain  23-2  x  0-51  =  n-8  cc.  of  the  AgNO3. 

So  20  -  11-8=8-2  cc.  of  the  AgNOs  have  been  precipitated. 
Now  i  cc   of  the  AgNO3  =  o-oi  gm.  NaCl, 
So  NaCl  in  10  cc.  urine  =  8-2  x  o-oi  gram. 
So  NaCl  in  100  cc.  urine  =  0-82  gram. 

413.    The  estimation  of  chlorides  by  Larrson's  method.* 

Principle.  The  pigments,  urates  and  other  interfering  substances  are 
removed  from  the  urine  by  adsorption  with  charcoal.  The  chlorides  are 
estimated  in  a  measured  amount  of  the  filtrate  by  direct  titration  with  silver 
nitrate,  using  potassium  chromate  as  an  indicator. 

Reagents  required. 

1.  Standard  silver  nitrate  (see  Ex.  412). 

2.  A  high  quality,  pure  absorbing  charcoal   (see  p.   390).     Ordinary 
animal  charcoal  is  quite  useless. 

3.  A  5  per  cent,  solution  of  potassium  chromate. 

*  Biochem.  Zeitschrift ,  xlix,  p.  479. 


354  ANALYSIS   OF   URINE.  [CH.  XIII. 

Method  of  analysis.  To  i  gram,  of  the  charcoal  in  a  dry  50  cc. 
flask  add  20  cc.  of  the  urine.  Shake  vigorously  and  repeat  the 
shaking  at  intervals  for  10  minutes.  Filter  through  a  small  dry 
paper  into  a  dry  tube.  Measure  10  cc.  of  the  nitrate  by  means  of  a 
pipette  and  transfer  it  to  a  small  beaker.  Add  5  or  6  drops  of  the 
chromate  and  titrate  with  the  standard  silver  nitrate  from  a  burette 
until  the  end  point  is  reached,  as  indicated  by  the  appearance  of  a 
reddish-brown  colour. 

Calculation.     I  cc.  of  silver  =  o-oi  gram.  NaCl. 

Example.     10  cc.  of  the  filtered  urine  required  10-6  cc.  of  silver. 
So  10  cc.  contain  10-6  x  o-oi  gram.  NaCl. 
So  100  cc.  contain  1-06  gram.  NaCl, 


J.     Phosphates. 
414.    The  estimation  of  phosphates. 

Principle.  Urine  is  heated  to  boiling  point,  and  titrated  whilst  hot  with  a 
standard  solution  of  uranium  acetate,  which  gives  a  precipitate  of  (UO2)HPO4 
with  phosphates  in  acetic  acid  solution.  Cochineal  tincture  is  used  to  indicate 
by  a  change  in  colour  when  the  uranium  is  in  excess. 

Reagents  required. 

1.  Acetate  solution.     Dissolve  100  grams,  of  sodium  acetate  in  a  litre  of 
distilled  water,  and  add  100  cc.  of  strong  acetic  acid. 

2.  Cochineal  tincture,  prepared  by  extracting  the  insects  with  30  per 
cent,  alcohol  and  filtering  after  two  days. 

3.  Standard  potassium  phosphate.     Dissolve  7-672  grams,  of  pure  re- 
crystallised  acid  potassium  phosphate  in  distilled  water  and  make  the  volume 
up  to  i  litre.    25  cc.  =  o-i  gram.  P2O6.    This  solution  can  also  be  prepared  by 
measuring  28-17  cc.  of  the  0-2  M.KH2PO4  described  on  page  24  into  a  TOO  cc. 
measuring  flask  and  making  the  volume  up  to  100  cc.  with  distilled  water. 

4.  Standard  uranium  acetate.     Dissolve  by  the  aid  of  heat  36  grams,  of 
pure  uranium  acetate  in  a  litre  of  distilled  water.     Allow  the  solution  to  cool 
and  then  filter.     Standardise  the  solution  as  follows  :    Into  a  beaker  measure 
25  cc.  of  the  standard  potassium  phosphate,  add  about  25  cc.  of  distilled 
water,  5  cc.  of  the  acetate  solution  and  about  i  cc.  of  the  cochineal  tincture. 
Bring  the  mixture  to  the  boiling  point  and  titrate  with  the  uranium  acetate 
solution  from  a  burette  till  the  red  tinge  just  changes  to  a  green,  heating  the 
mixture  to  boiling  just  before  the  last  few  drops  are  added.     If  x  cc.  of  the 
uranium    solution    are   used,   then    i    cc.    of  the   uranium    corresponds    to 

o-i 

—  gram.  P2O6. 

If  desired  the  solution  can  be  diluted  with  water  so  that  i  cc.  =  0-005  gram. 
PaO6.  To  effect  this  add  —  -  cc.  of  distilled  water  to  every  100  cc. 


CH.  XHI.J  SULPHATES.  355 

Method.  In  a  beaker  of  about  100  cc.  capacity  place  50  cc. 
urine,  add  5  cc.  of  the  sodium  acetate  solution  and  about  I  cc.  of  the 
cochineal  tincture.  Have  a  burette  ready  containing  the  stand- 
ardised uranium  acetate  solution.  Heat  the  urine  to  boiling 
point,  remove  the  flame  and  run  in  the  uranium  acetate  as  long  as 
a  precipitate  is  formed.  Heat  the  mixture  again  just  to  boiling 
point,  and  cautiously  add  uranium  acetate,  drop  by  drop,  till  the 
red  colour  is  converted  to  a  green. 

Calculation. 

i  cc.  of  the  uranium  acetate  =  0-005  gram.  P2O5. 

Thus  if  50  cc.  of  urine  require  15-2  cc.  uranium,  the  percentage  of  P2O5  is 
2  x  15-2  x  0-005  =  0-152  gram. 

K.     Sulphates. 

Sulphates  can  be  determined  gravimetrically  as  barium  sulphate  or 
volumetrically  by  means  of  benzidine.  The  latter  is  much  more  convenient, 
but  both  methods  are  given  below.  The  difficulty  encountered  with  the 
gravimetric  method  is  that  of  preventing  adsorption  of  other  substances.  For 
that  reason  the  barium  must  be  added  very  slowly  and  the  method  is  extremely 
tedious. 

415.    The  estimation  of  total  sulphates  by  Folin's  method.* 

Place  25  cc.  of  urine  in  a  250  cc.  Erlenmeyer  flask,  add  20  cc. 
of  hydrochloric  acid  (i  volume  of  concentrated  HC1  to  4  volumes  of 
water)  and  boil  gently  for  30  minutes,  covering  the  mouth  of  the 
flask  with  a  small  watch  glass.  Cool  the  flask  under  the  tap  and 
dilute  to  about  150  cc.  with  water.  Add  10  cc.  of  5  per  cent,  barium 
chloride  solution  slowly,  drop  by  drop,  to  the  cold  solution,  which 
must  not  be  stirred  or  shaken  during  the  addition,  nor  for  at  least 
one  hour  after.  Then  shake  well,  filter  through  a  weighed  Gooch 
crucible  (see  note  to  Ex.  410),  wash  with  250  cc.  of  cold  water,  dry 
in  an  air  bath,  or  over  a  very  low  flame.  Ignite,  cool  and  weigh. 

Calculation.     Weight  of  BaSO4  x  i  -366  =  SO8  per  cent. 

NOTES. — Instead  of  using  a  Gooch  crucible  a  washed  "  Barium  sulphate  " 
filter  paper  may  be  used. 

After  washing  and  drying  the  ignition  may  be  carried  out  in  a  platinum  or 
porcelain  crucible,  previously  weighed.  After  ignition,  the  ash  should  be 
treated  with  a  drop  of  25  per  cent,  sulphuric  acid,  cautiously  dried  and  heated 
again. 

A  correction  must  be  made  for  the  weight  of  the  ash  of  the  paper. 

*  Journ.  Biol.  Chem.,  i.,  p.  150. 


356  ANALYSIS   OF   URINE.  [CH.  XIII. 

416.  The  estimation  of  inorganic  sulphates  by  Folin's  method. 

Place  25  cc.  of  urine  and  100  cc.  of  water  in  a  250  cc.  Erlenmeyer 
flask.  Acidify  with  10  cc.  of  hydrochloric  acid  (i  volume  of  con- 
centrated HC1  to  4  volumes  of  water).  Add  10  cc.  of  5  per  cent, 
barium  chloride,  drop  by  drop,  as  in  the  previous  exercise,  and 
proceed  as  there  directed. 

Calculation.     The  same  as  for  total  sulphates. 

Ethereal  Sulphates. 

This  can  be  found  by  difference.  Total  sulphates  less  inorganic 
sulphates  =  ethereal  sulphates. 

417.  The  estimation  of  total  sulphur  by  Benedict's  method.* 

Place  10  cc.  of  urine  in  a  small  (7-8  cm.)  porcelain  or  silica 
crucible  and  add  5  cc.  of  Benedict's  sulphur  reagent.  Evaporate 
over  a  free  flame,  keeping  the  solution  just  below  the  boiling  point, 
to  prevent  loss  by  spattering.  When  dry,  raise  the  flame  slightly 
until  the  entire  residue  has  blackened.  Raise  the  flame  still  more 
and  heat  to  redness  for  ten  minutes  after  the  black  residue  (which 
first  fuses)  has  become  dry.  Allow  the  dish  to  cool.  Add  10  to  20 
cc.  of  i  in  4  hydrochloric  acid,  and  heat  again  till  the  residue  has 
completely  dissolved  to  a  clear  solution.  Wash  the  contents 
quantitatively  into  an  Erlenmeyer  flask,  and  dilute  with  cold  water 
to  100  to  150  cc.  Add  10  cc.  of  10  per  cent,  barium  chloride,  drop 
by  drop,  and  allow  to  stand  for  about  an  hour.  Shake  thoroughly 
and  proceed  as  in  Ex.  415. 

Calculation.  Weight  of  BaSO4  from  10  cc.  of  urine  multiplied  by  3*413 
=  SO8  per  cent. 

NOTE.     Benedict's  sulphur  reagent  is  : 

Crystallised  copper  nitrate,  200  grams. 
Potassium  chlorate,  50  grams. 
Distilled  water  to  i  litre. 

Neutral  Sulphur. 

This  can  be  found  by  difference.  Total  sulphates  less  total 
sulphur  =  neutral  sulphur. 

*  Journ.  Biol.  Chem.,  vi.,  p.  363. 


CH.  XIII.]  SULPHATES.  357 

418.  Inorganic  sulphates  by  the  benzidine  method  of 
Rosenheim  and  Drummond.* 

Principle.  The  urine  is  acidified  with  hydrochloric  acid  and  treated  with 
an  excess  of  benzidine  hydrochloride.  The  sulphates  are  precipitated  quantir 
tatively.  The  precipitate  is  filtered  off  under  suction,  washed  free  from  acid 
with  water  (or  better  with  a  saturated  solution  of  benzidine  sulphate)  and 
suspended  in  hot  water.  Phenol  phthalein  is  added,  and  the  mixture  titrated 
with  standard  soda.  A  pink  colour  does  not  develop  until  enough  soda  has 
been  added  to  combine  with  the  whole  of  the  benzidine  sulphate  to  form 
sodium  sulphate.  Benzidine  sulphate,  being  the  salt  of  a  weak  base  with  a 
strong  acid,  suffers  hydrolytic  dissociation  into  the  base  and  the  acid.  The 
base  is  only  very  feebly  ionised,  whilst  the  strong  acid  is  freely  ionised, 
the  solution  in  hot  water  thus  behaving  like  sulphuric  acid,  which  can  be 
titrated  with  the  standard  soda. 

Solutions  required. 

1.  Benzidine  hydrochloride.     Rub  up  4  grams,  of  pure  benzidine  with 
about  10  cc.  of  distilled  water.     Transfer  with  about  500  cc.  of  water  to  a  2 
litre  flask.     Add  5  cc.  of  concentrated  hydrochloric  acid  and  make  up  to  2 
litres  with  distilled  water. 

2.  Hydrochloric  acid.     Dilute   i   volume  of  pure  concentrated  hydro- 
chloric acid  with  3  volumes  of  distilled  water. 

3.  Saturated  benzidine  sulphate.     Prepare  some  benzidine  sulphate  by 
adding  a  little  sodium  sulphate  to  200  cc.  of  the  benzidine  hydrochloride. 
Collect  the  precipitate  as  described  below  and  wash  it  thoroughly  with  cold 
water.     Suspend  it  in  a  considerable  volume  of  hot  water  and  allow  it  to 
stand  over-night  in  a  cool  place.     Filter  from  the  benzidine  sulphate  till  quite 
clear. 

4.  o-i   N.   sodium  hydroxide.     See  appendix.     The  exact  strengthens 
immaterial,  so  long  as  it  be  accurately  determined. 

5.  Phenol  phthalein.     A  saturated  solution  in  alcohol. 

Method.  Measure  25  cc.  of  the  urine  (filtered,  if  necessary) 
into  a  250  cc.  Erlenmeyer  flask,  with  a  wide  neck.  Add  2  cc.  of  the 
hydrochloric  acid  and  100  cc.  of  the  benzidine  hydrochloride.  Mix 
and  allow  to  stand  for  10  minutes.  Filter  through  paper  and  paper 
pulp,  as  described  in  Ex.  406.  The  filtrate  must  be  crystal  clear. 
If  it  is  cloudy  it  must  be  passed  through  the  filter  again.  Wash  out 
the  beaker  with  10  cc.  of  the  saturated  benzidine  sulphate  and  wash 
the  precipitate  with  this.  Repeat  this  at  least  once  more.  Transfer 
the  precipitate,  filter  pulp  and  disc  to  the  Erlenmeyer  flask  and  wash 
the  funnel  into  the  flask  with  a  jet  of  boiling  water,  using  about 
50  cc.  of  water.  Any  lumps  of  the  precipitate  must  be  broken  up 
by  use  of  a  glass  rod  before  the  titration  is  commenced,  or,  if  this  is 

*  Biochemical  Journal,  viii.,  p.  134. 


358 


ANALYSIS    OF  URINE. 


[CH.  XIH. 


impossible,  before  the  titration  is  completed.     Add  a  few  drops  of 
the  saturated  solution  of  phenol  phthalein  and  titrate  the  hot  solu- 
tion with  the  standard  soda.    The  end  point  is  quite  sharp. 
Calculation,     i  cc.  of  o-i  N.  soda  iff  4-0  mg.  SO8. 

419.  Total  sulphates  by  the  benzidine  method.  Measure 
25  cc.  of  the  urine  into  the  Erlenmeyer  flask,  add  2  to  2*5  cc.  of  the 
hydrochloric  acid  and  20  cc.  of  distilled  water.  Place  a  funnel  in 
the  neck  of  the  flask  and  boil  gently  for  20  minutes.  Cool  thoroughly 
under  the  tap,  add  100  cc.  of  the  benzidine  hydrochloride,  and 
proceed  as  directed  above. 

Ethereal  Sulphates. 

The  difference  between  the  result  of  the  analysis  in  Ex.  419  and 
that  of  Ex.  418  is  the  ethereal  sulphate. 

Total  Sulphur  and  Neutral  Sulphur. 

The  urine  can  be  oxidised  by  Benedict's  method  (Ex.  417)  and 
the  residue  dissolved  in  hydrochloric  acid.  Before  the  benzidine 
method  is  applied  the  excess  of  free  hydrochloric  acid  must  be  reduced 
by  the  addition  of  soda  until  the  solution  is  only 
just  acid  to  congo  red  paper.  The  calculation 
for  neutral  sulphur  is  explained  in  Ex.  417. 

L.    Albumin. 

420.  The  estimation  of  albumin  by 
Esbach's  method. 

Fill  the  albuminometer  to  the  mark  U 
with  urine.  Add  Esbach's  reagent  (Ex.  14) 
to  the  mark  R.  Stopper  the  tube,  and  invert 
it  slowly  several  times  to  mix  the  fluids.  Allow 
the  tube  to  stand  upright  for  24  hours. 

Calculation.  The  graduations  on  the  albumino- 
meter indicate  grams,  of  albumin  per  litre. 


Fig.  47.      Esbach's 
albuminometer. 


421.    The  estimation  of  albumin  by  Scherer's  method. 

Measure  50  cc.  of  urine  into  a  beaker.    Place  it  on  a  water  bath 
and  raise  the  temperature  to  50°  C.    Add  i  per  cent,  acetic  acid, 


CH.  xm.] 


DIASTASE. 


359 


drop  by  drop,  to  obtain  a  complete  separation  of  the  protein  (care 
must  be  taken  to  avoid  an  excess) .  Raise  the  temperature  to  boiling 
and  keep  it  so  for  a  few  minutes.  Filter  the  urine  through  a  small 
paper  that  has  previously  been  washed,  dried  and  weighed.  Wash 
the  precipitate  in  turn  with  hot  water,  95  per  cent,  alcohol  and  ether. 
Dry  the  paper  and  precipitate  in  an  air  bath  at  110°  C.  till  the  weight 
is  constant.  The  weight  of  protein  in  50  cc.  is  obtained  by  subtract- 
ing the  weight  of  the  paper. 

M.    Diastase. 
422.    Wohlgemuth's  method. 

Principle.  Varying  amounts  of  urine  are  added  to  a  given  amount  of 
soluble  starch,  and  the  mixture  digested  for  30  minutes  at  38°  C.  After  cool- 
ing, a  drop  of  dilute  iodine  is  added  to  each  tube.  The  tubes  that  contain 
considerable  amounts  of  urine  have  all  the  starch  digested  so  that  no  colour 

1  s  obtained  on  adding  the  iodine.     The  tube  with  the  smallest  amount  of  urine 
that  completely  digests  the  starch  is  found  and  so  the  diastatic  value  calcu- 
lated (see  p.  192). 

Reagents  required. 

1.  Stock  solution  of  soluble  starch.     Accurately  weigh  out  2  grams,  of 
soluble  starch  (see  p.  391)  and  transfer  it  to  a  dry  test-tube.     Add  about 
10  cc.  of  distilled  water  and  shake.     Pour  the  suspension  into  about  70  cc.  of 
boiling  distilled  water  and  stir  well.  Wash  the  tube  three  successive  times  with 
5  cc.  of  distilled  water,  transferring  the  washings  to  the  boiling  solution.     Now 
add  10  grams,  of  pure  sodium  chloride.     Allow  to  cool  and  make  the  volume 
up  to  100  cc.  with  distilled  water.     The  solution  is  stable  for  months. 

2.  One  per  mille  soluble  starch  in  0-5  per  cent,  sodium  chloride.     5  cc. 
of  the  stock  are  diluted  with  distilled  water  to  make  100  cc.     This  solution 
must  be  freshly  prepared  each  day. 

3.  N/50  iodine,  prepared  from   N/io  iodine   (see  p.  390)  by  diluting 

2  cc.  with  8  cc.  of  distilled  water.     The  diluted  iodine  must  be  freshly  prepared 
each  day. 

Method.    Label  a  series  of  clean  dry  test-tubes  i  to  10. 
Into  the  tubes  measure  the  volume  of  urine  and  of  distilled 
water  stated  in  the  table,  using  guarded  pipettes  (see  Note  7). 


Tube. 

c.c.  of  Urine. 

c.c.  of 
Water. 

i*2§! 
30' 

Tube. 

c.c  of  Urine 
diluted  1  in  10 
with  water. 

c.c.  of 
Water. 

d™l 

30' 

1 

0-5 

0-5 

4 

6 

0-9 

0-1 

22-2 

2 

0-4 

0-6 

5 

7 

0-8 

0-2 

25 

3 

0-3 

0-7 

6-6 

8 

0-7 

0-3 

28-6 

4 

0-2 

0-8 

10 

9 

0-6 

0-4 

33-3 

5 

o-i 

0-9 

20 

10 

0-5 

0-5 

40 

360  ANALYSIS  OF  URINE.  [CH.  xm. 

To  each  tube  add  2  cc.  of  the  one  per  mille  starch,  commencing 
with  tube  10.  Mix  the  contents  by  agitation  and  place  in  a  thermo- 
stat or  a  water  bath  at  38°  C.  for  exactly  30  minutes. 

Remove  the  tubes  and  place  them  in  a  beaker  of  cold  water  for 
3  minutes  to  cool. 

Arrange  the  tubes  in  order  in  a  stand. 

Commencing  with  tube  I  add  I  drop  of  the  N/5O  iodine  to  each 
tube  and  carefully  note  the  colour  produced. 

Should  a  colour  be  produced  and  it  rapidly  fades,  add  i  more 
drop  of  iodine  to  each  tube. 

Note  the  tube  with  the  lowest  number  that  shows  a  blue  tinge. 
The  next  lower  tube  contains  an  amount  of  urine  that  completely 
digests  2  cc.  of  o-i  per  cent,  starch  in  30  minutes  at  38°  C. 

Calculation.  This  is  explained  on  page  192.  The  d  values  corresponding 
to  the  volumes  of  urine  required  are  given  in  the  table.  Thus  if  tube  4  shows  a 
bluish  tint  and  tube  3  a  red,  then  d—  6-6. 

NOTES. — i.  It  is  customary  to  use  a  freshly  prepared  o«i  per  cent, 
solution  of  starch  in  water  and  to  make  the  volume  of  the  urine  up  to  i  cc. 
with  i  per  cent,  sodium  chloride.  The  author  has  determined  that  2  per  cent, 
starch  in  10  per  cent,  sodium  chloride  is  quite  stable  and  that  the  results 
obtained  agree  with  these  found  by  the  original  method.  It  is  suggested 
that  the  present  more  convenient  method  be  adopted  as  a  standard. 

38° 

2.  The  d — 7  of  normal  urine  varies  between  5  and  20,  with  an  average 

of  10.     In  acute  pancreatitis  the  value  is  high  and  may  be  over  200.     In  such 
cases  the  urine  is  still  further  diluted  to  i  in  100  and  the  d  calculated. 

2 

Thus  if  O'Oo6  cc.  of  urine  is  required  d  =        ^  =  333. 

3.  It  is  important  that  the  same  amount  of  iodine  be  added  to  each  tube. 
Very  uneven  results  are  obtained  if  varying  amounts  of  iodine  be  employed. 

4.  Samples  of  the  mixed  24  hours'  specimen  should  be  used. 

5.  The  diastase  in  the  urine  is  quite  stable  if  the  urine  be  preserved  by 
the  addition  of  toluol.     3  cc.  are  ample  for  an  estimation. 

6.  The  pipettes  for  measuring  the  solutions  must  be  accurate  i  cc. 
pipettes  graduated  to  i/ioo  cc. 

7.  The  end  of  the  pipettes  that  are  placed  in  the  mouth  should  be  guarded 
by  plugs  of  cotton  wool,  to  prevent  contamination  of  the  fluids  with  saliva. 
Bewildering  results  in   class  work  disappeared  after  this  precaution   was 
rigorously  enforced. 


CHAPTER   XIV. 

DETECTION  OF  SUBSTANCES  OF  PHYSIOLOGICAL 

INTEREST. 

If  no  indication  as  to  the  origin  of  the  substance  is 
available  the  scope  of  the  analysis  is  very  considerable. 
The  following  account  is  not  intended  to  be  exhaustive, 
but  merely  to  suggest  a  few  methods  of  attack.  Success 
demands  a  sound  knowledge  of  the  properties  and  reactions 
of  a  large  number  of  substances.  Experience,  practice  and 
enterprise  count  for  a  good  deal.  Many  substances  are 
not  detected  by  student  analysts  mainly  because  they 
forget  to  test  for  them.  The  hints  on  page  368  should  be 
carefully  studied.  The  student  is  urged  to  perform  his 
tests  on  the  smallest  amount  of  material  that  is  likely  to 
give  a  conclusive  result.  With  a  limited  supply  of  the 
substance  for  analysis  a  much  greater  variety  of  tests  can 
thus  be  applied. 

A.    Fluids. 

1.  A  portion  may  be  neutralised  and  evaporated  to  dryness  on 
the  water  bath.    This  allows  for  a  subsequent  extraction  with  strong 
alcohol,  which  serves  for  the  separation  of  many  substances.     It 
should  not  be  started  until  there  is  some  indication  that  it  may  be 
necessary,  as  for  the  separation  of  sugars  from  proteins  and  poly- 
saccharides,  etc.     The  evaporation  must  be  conducted  in  neutral 
solution  to  obviate  any  chemical  changes  produced  by  hot  acids  or 
alkalies. 

2.  Note  any  characteristic  smell  of  urine,  bile,  etc.     In  such 
cases  apply  tests  for  characteristic  constituents. 

3.  Note  the  colour  and  appearance  of  the  fluid :    opalescence 
suggests  starch,  glycogen,  or  certain  protein  solutions;    coloured 
fluids  suggest  bile,  blood  or  urine. 


DETECTION  OF  SUBSTANCES.  [CH.  XIV. 

4.  Note  the  reaction  to  litmus.     An  acid  reaction  excludes 
the  presence   of  mucin,   nucleoproteins,   caseinogen,  and  usually 
earthy  phosphates. 

5.  If  acid  test  for  free  HC1  by  Gunsberg's  test.     (Ex.  246.) 

6.  Sprinkle  some  flowers  of  sulphur  on  the  surface  of  a  portion 
of  the  fluid  in  a  test-tube.     If  the  particles  fall  through  the  surface, 
bile  salts  are  probably  present.     (Ex.  316.)     Confirm  by  Petten- 
kofer's  test.     (Ex.  315.) 

7.  If  the  fluid  be  brown  or  green,  apply  the  Huppert-Cole  test 
(Ex.  318)  for  bile  pigments. 

8.  If  the  fluid  be  red  or  brown,  examine  for  blood-pigments 
or  derivatives  by  Table  F,  page  366. 

9.  If  there  are  any  reasons  for  suspecting  the  presence  of 
ferments,  examine  as  directed  on  page  367.     If  none  of  the  colour 
reactions  for  proteins  are  obtained,  ferments  are  probably  absent. 

10.  Examine  for  proteins  by  Millon's  and  the  biuret  reactions 
(Ex.  22  and  24).     If  they  be  present,  proceed  as  directed  in  Table 
A,  B  or  C,  according  to  the  reaction  of  the  fluid. 

11.  If  proteins  are  absent,  proceed  to  Table  E. 

12.  Test  for  uric  acid  if  the  fluid  be  alkaline,  neutral  or  only 
faintly  acid.     Acidify  with  a  drop  or  two  of  strong  hydrochloric 
acid;  uric  acid  may  separate  out  as  a  crystalline  powder.     Make 
another  portion  of  the  solution  alkaline  with  ammonia,  saturate 
with  NH4C1  and  apply  the  murexide  reaction  to  the  precipitate  thus 
obtained.     (Ex.  352.) 

13.  If  the  fluid  be  alkaline,  treat  a  little  with  a  solution  of 
calcium  chloride.    A  white  curdy  precipitate  indicates  the  presence 
of  soaps.     (Their  presence  should  be  confirmed  by  the  methods 
given  in  Ex.  177.) 

14.  Treat  a  portion  with  a  little  hypobromite  solution.     If  an 
effervescence  is  obtained,  test  for  urea  by  Ex.  343.     If  this  is 
negative  the  solution  may  contain  ammo-acids  or  ammonium  salts. 
The  bromine  test  for  free  tryptophane  (p.  217)  may  give  a  valuable 
indication. 


CH.  XIV.] 


PROTEINS. 


363 


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DETECTION   OF  SUBSTANCES. 


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CH    XIV.]  NON-COAGULABLE  PROTEINS.  365 

Table  D. 

Examination  of  Filtrate  B  for  albumoses,  peptones  and  gelatin. 


Treat  a  portion  with  caustic  soda  and  a  drop  of  copper  sulphate  solution. 

No  biuret 

Positive  biuret  reaction.     To  portions  of  filtrate  B  apply 

reaction. 

Millon's  and  glyoxylic  tests. 

Negative 

Positive   reactions.     Saturate  nitrate  B  with 

reactions. 

ammonium  sulphate  by  heating  with  excess  of 

solid.     Cool  under  tap  and  filter. 

Gelatin 

present. 

Confirm  by 

Precipitate.     Mostly   stick- 
ing  to   tube.      Wash    with 

Filtrate. 
Treat     2     cc. 

obtaining  a 

cold    saturated    ammonium 

with    4  cr    of 

precipitate 
by      half- 

sulphate.       Dissolve     in     a 
little  boiling  water  and  cool 

40    per    cent. 
NaOH   and  a 

saturation 
with   amm. 
sulphate. 

under     tap.      To    portions 
apply  biuret  test  (using    40 
per  cent.  NaOH)    and   the 

drop  of  copper 
sulphate. 
Pink      colour 

glyoxylic  test.     If  both  are 

indicates 

positive  — 

Proteins 

Albumoses. 

Peptones. 

absent. 

Table   E. 

Examination  of  a  solution  for  carbohydrates. 

If  proteins  be  present  they  must  be  removed,  as  far  as  possible, 
by  neutralising,  boiling  and  filtering. 

In  any  case  the  solution  tested  must  be  neutral. 

(a)  To  a  small  portion  add  diluted  iodine  drop  by  drop,  until 
an  excess  has  been  added.  If  a  pure  blue  colour  be  obtained  at  any 
stage  of  the  addition  of  iodine,  starch  is  present.  If  a  purple  or 
brown  colour  be  produced  and  the  fluid  be  quite  clear,  erythro- 
dextrin  is  present  and  glycogen  absent.  If  a  blue  colour  be 
produced,  or  if  the  fluid  be  opalescent,  proceed  as  follows : 


366 


DETECTION   OF  SUBSTANCES. 


[CH.  XIV. 


To  a  portion  of  the  fluid,  prepared  as  directed  above,  add  an 
equal  bulk  of  saturated  (NH4)2SO4,  shake  vigorously,  and  filter 
through  a  dryjpaper  after  about^ten  minutes. 


Precipitate. 
Scrape  off  the  paper, 
dissolve  in  a  little  hot 
water,  cool  and  add  a 
drop  of  iodine.  A  blue 
colour  shows  the  pres- 
ence of  starch. 


Filtrate.  To  a  small  portion  add  a  drop  or  two 
of  iodine.  If  a  reddish  or  purple  colour  be  produced, 
glycogen  or  dextrin  is  present.  If  the  fluid  be 
opalescent  after  warming,  glycogen  is  present. 
Saturate  the  remainder  with  (NH<)2SO4  and  filter. 


Precipitate. 
Neglect. 


Filtrate.  Add  a  drop  of  diluted 
iodine,  a  red-brown  colour  shows 
the  presence  of  erythro-dextrin, 


(b)  Apply  Benedict's  (Ex.  TOO)  or  Fehling's  test  (Ex.  97)  for 
reducing  sugars.     Note  that   the  tests  do  not  succeed  in  the 
presence  of  any  considerable  amount  of  ammonium  salts.    Also  that 
if  albumoses,    peptones    or  gelatin  are  present  they  should  be 
removed  by  alcoholic  extraction  as  described  in  Ex.  58. 

(c)  If  a  reduction  be  obtained,  apply  Barfoed's  test  (Ex.  101) 
to  distinguish  between  mono-  and  di-saccharides.    The  osazone  test 
(Ex.  109)  also  can  be  applied  if  necessary. 

(d)  Test  for  cane-sugar  and  fructose  (see  Exs.  130  and  131). 


Table  F. 

Examine  the  solution  spectroscopically :  gradually  dilute  the 
solution,  noting  the  spectrum  at  all  stages  of  dilution. 

Take  the  reaction  of  the  undiluted  fluid  to  litmus  paper,  wash- 
ing the  surplus  off  the  paper  with  a  stream  of  distilled  water,  if  you 
are  unable  to  note  the  reaction  directly. 

If  the  fluid  be  neutral  or  alkaline,  treat  it  with  Stokes'  fluid  or 
warm  it  with  ammonium  sulphide,  and  note  whether  the  spectrum 
is  altered  by  reduction.  This  should  be  done  after  various  dilutions 
of  the  original  solution. 


CH.  XIV. 


Fluid  red 


PIGMENTS   AND   ENZYMES. 
Acid  —  Acid  haematoporphyrin,  two  bands.    (Ex.  307.) 


36; 


Neutral 


lAlkaline 


Dilute  till  two 

bands  are  well 

seen  and  then 

reduce. 


Oxy haemoglobin,  the  two  bands 
merge  into  one  faint  band  (Ex 
293.) 

CO -haemoglobin,  the  two  bands 
are  unaltered.  (Ex.  296.) 


Alkaline  haematoporphyrin,  feur  bands,,  converted 
into  acid  haematoporphyrin  by  strong  acids.  (Exs. 
307  and  308.) 

Haemochromogen,  two  bands  in  green,  one  much 
more  distinct  than  the  other,  unaffected  by  reduc- 
ing reagents.  (Ex.  305.) 


Fluid  brown 


Acid — Acid  haematin,  band  in  red.        Ex.  301., 

Neutral.  Methaemoglobin,  band  in  red  :  gives  spectrum  of 
oxy haemoglobin  and  then  of  reduced  haemoglobin 
if  reduced.  (Ex.  299.) 

Alkaline  haematoporphyrin — four  bands.     (Ex.  308.) 


VAlkaline  - 


Alkaline  haematin,  faint  band  in  red,  converted  to 
haemochromogen  by  reducing  reagents.  (Exs. 
303,  305.) 


Enzymes. 

In  testing  for  enzymes  it  is  important  to  note  the  reaction.  "s!\It  is  not 
necessary  to  determine  the  exact  PH,  but  trials  should  be  made  with  litmus, 
followed  by  phenol-red  and  phenol-phthalein  for  alkaline  solutions,  and 
methyl-red  and  brom-phenol-blue  (or  thymol-blue)  for  acid  solutions.  In  this 
way  certain  valuable  indications  may  be  obtained. 

The  next  point  to  remember  is  that  all  tests  must  be  made  in  parallel  with 
a  control.  In  the  control  test,  the  solution  is  well  boiled  to  destroy  any 
enzyme  that  may  be  present :  in  other  respects  it  is  carried  out  exactly  as  the 
test  proper.  Without  this  precaution  it  is  quite  impossible  to  make  any  safe 
deduction. 

To  test  for  proteolytic  enzymes  see  if  the  solution  will  clot  calcined  milk 
(Exs.  252  and  257) .  The  solution  should  be  nearly  neutralised  before  applying 
the  test,  but  if  it  is  acid  it  must  not  be  made  alkaline  (see  Ex.  253).  If  this 
test  is  positive,  pepsin  can  be  identified  by  the  method  given  in  Ex.  248. 
Rennin  can  be  distinguished  from  pepsin  by  Ex.  256,  though  it  takes  a  good 
deal  of  time.  It  is  unusual  to  find  a  rennin  solution  free  from  pepsin,  but  many 
commercial  pepsins  are  practically  free  from  true  rennin.  Trypsin  can  be 
identified  by  Ex.  258. 


368  DETECTION   OF   SUBSTANCES.  [CH.  XIV. 

Diastatic  enzymes  are  probably  absent  if  the  solution  is  strongly  acid 
or  alkaline.  The  reaction  and  salt  content  are  factors  of  importance.  The 
best  method  of  testing  for  these  enzymes  is  some  modification  of  Ex.  237, 
adding  the  buffer  and  the  salt  for  the  reasons  given  in  that  exercise.  The 
solution  should  be  carefully  neutralised  to  litmus  before  making  the  tests. 

The  enzymes  acting  on  the  disaccharides  are  usually  more  difficult  to 
identify.  Sucrase  is  sometimes  found  in  a  very  active  condition,  but  the  tests 
for  maltase  and  lactase  generally  require  an  incubation  period  of  at  least 
15  hours.  For  details  see  Exs.  266-268. 

Lipase  is  rather  unstable  to  acids.     For  tests  see  Ex.  168. 


A  few  special  hints  on  the  examination  of  physiological  fluids. 

1.  It  is  impossible  to  obtain  a  heat  coagulum  of  albumin  or 
globulin  in  an  acid  or  alkaline  fluid.     The  reaction  must  be  neutral 
or  only  very  faintly  acid. 

2.  A  little  litmus  solution  in  the  fluid  does  no  harm,  and  often 
reminds  one  that  the  reaction  changes  after  boiling  (owing  to  the 
evolution  of  CO2). 

3.  In  testing  for  peptones,  after  removing  the  albumoses  by 
saturation  with  ammonium  sulphate,  the  biuret  test  succeeds  only  if 
at  least  two  volumes  of  40  per  cent,  soda  are  used.     The  test  will  not 
be  obtained  with  the  ordinary  5  per  cent,  soda  (see  notes  to  Ex.  57). 

4.  Gelatin  reacts  very  much  like  the  albumoses,  except  that 
it  does  not  yield  the  glyoxylic  reaction.     It  can  be  precipitated  by 
half-saturation  with  ammonium'  sulphate.     If  the  precipitate  is 
collected,  squeezed  and  dissolved  in  a  very  little  hot  water,  the  solu- 
tion will  often  set  after  being  thoroughly  cooled  for  some  time. 

5.  It  is  impossible  to  obtain  Fehling's  or  Benedict's  test  for 
the  reducing  sugars  in  the  presence  of  any  considerable  amount  of 
ammonia  or  ammonium  salts. 

6.  The  sugars  reduce  only  in  an  alkaline  medium.     If  the 
fluid  under  examination  be  acid,  it  must  be  neutralised  before 
boiling  with  the  Fehling's  or  Benedict's  solution. 

7.  In  testing  for  cane  sugar  do  not  forget  that  starch  and  the 
dextrins  are  hydrolysed  to  glucose  by  boiling  acids.    But  whereas 


CH.  XIV.]  ANALYSIS   OF   FLUIDS.  369 

cane  sugar  is  hydrolysed  very  easily,  starch,  etc.,  are  only  slowly 
acted  on. 

8.  Starch,  glycogen  and  the  erythro-dextrins  do  not  give  any 
colour  with  iodine  solutions,  if  the  reaction  of  the  fluid  be  alkaline. 
If  this  be  the  case,  make  the  reaction  acid  with  acetic  acid. 

9.  The  proteins  interfere  with  the  iodine  tests  for  these 
substances,  and  should  therefore  as  far  as  possible  be  removed 
before  testing  for  the  polysaccharides. 

10.  Fat  is  insoluble  in  water,  so  do  not  waste  time  in  testing 
an  ordinary  solution  for  fats. 

11.  The  only  reliable  test  for  urea  is  the  urease  test  (Ex.  343). 
In  this  connection  it  must  be  remembered  that  urea  is  soluble  in 
alcohol,  and  can  thus  be  separated  from  the  proteins  and  other 
substances  likely  to  interfere  owing  to  their  "  buffer  "  action. 

12.  Ammonium  chloride  is  a  very  valuable  reagent  in  testing 
for  uric  acid  or  urates.    The  only  other  physiological  substance 
precipitated  by  it  is  soap. 

13.  Never   omit    "  control "   tests   when   investigating   the 
ferment  action  of  a  solution. 

14.  Use  "  carmine  fibrin  "  in  testing  for  pepsin  ;  never  when 
testing  for  trypsin. 

15.  In  testing  solutions  for  pigments,  examine  spectroscopi- 
cally  in  various  dilutions.     Note  the  reaction  of  the  fluid ;  it  is 
no  good  looking  for  haemochromogen  in  a  markedly  acid  solution. 

16.  Creatinine,  acetone,  aceto-acetic  acid  and  lactic  acid  can 
usually  be  identified  by  specific  colour  reaction,  though  the  latter 
generally  involves  an  extraction  with  ether.     Creatine  can  only  be 
identified  after  conversion  to  creatinine,  and  then  an  estimation  of 
total  creatinine  is  necessary. 

17.  A   solution  of  amino-acids  evolves  nitrogen  gas  with 
nitrous  acid  and  also  with  alkaline  hypobromites.     Ammonia  can 
be  removed  by  gentle  boiling  in  an  open  dish  with  a  little  alkali. 

AA 


37O  DETECTION    OF   SUBSTANCES  [CH.  XIV. 

B.    Solids. 

1.  Examine  a  little  microscopically,  both  dry  and  with  the 
addition  of  a  drop  of  water.     Look  for  starch  grains,  crystals  of 
urea,  uric  acid,  urates,  leucine,  tyrosine,  cholesterol,  and  haemin 
scales. 

2.  Heat  a  small  amount  of  the  solid  in  a  dry  tube,    at   first 
gently  and  then  more  strongly. 

(a)  If  sublimation  take  place  and  an  odour  of  amylamine  be 
given  off,  leucine  is  present. 

(b)  If  sublimation  take  place  and  a  strong  smell  of  ammonia 
be  evolved,  urea  is  indicated. 

(c)  A  smell  of  phenol  and  nitro-benzol  indicates  tyrosine. 

(d)  A  smell  of  burning  feathers  indicates  proteins,  gelatin,  etc. 

(e)  A  smell  of  acrolein  indicates  fats. 

3.  Boil  some  of  the  solid  with  a  small  amount  of  water  in 
a  tube,  cool  under  the  tap  and  leave  the  test-tube  in  a  beaker 
of  cold  water  for  10  minutes.     If  gelatin  be  present,  the  solution 
will  set  to  a  jelly.     (Starch,  if  present,  may  form  a  thick  paste, 
which  may  be  confused  with  the  clean  jelly  given  by  gelatin.     If 
the  tube  be  subsequently  placed  in  boiling  water,  gelatin  becomes 
quite  limpid,  whilst  starch  remains  thick.) 

4.  If  the  solid  be  of  a  dark  brown  or  red  colour,  boil  a  portion 
with  dilute  alkali,  filter,  heat  the  filtrate  with  Stokes'  fluid  or 
ammonium  sulphide,  and  examine  for  the  spectrum  of  haemochro- 
mogen.     (Ex.  305.)     If  this  be  obtained,  the  solid  contains  dried 
blood  or  haematin.     Confirm  by  obtaining  haemin  crystals.     (Ex. 
309-) 

5.  The  table  on  the  next  page  can  be  followed,  but  the  method 
adopted  will  depend  on  the  indications  obtained  by  preliminary 
tests.     It  is  advisable  to  test  for  starch  before  deciding  on  a  plan  of 
operation. 


CH.  XIV.] 


SOILDS. 


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APPENDIX. 


i  grain  =  -0648  gram. 

i  ounce  =  437-5  grains  =  28-3595  grams. 

i  Ib.  =  1 6  oz.  =  7000  grains  =  453-5925  grams. 

i  gram.  =  15-432  grains. 

i  kilogram  =  1000  grams  =  2-2046  Ibs. 

i  minim  =  -059  cc. 

i  fluid  drachm  =  60  minims  =  3-55  cc. 

i  fluid  ounce  =  8  fluid  drachms  =  28-4  cc. 

i  pint  =  20  fluid  oz.  =  567-9  cc. 

i  cc.  =  16-9  minims. 

i  litre  =  1000  cc.  =  35-2  fluid  oz.  =  1-76  pints. 

i  gallon  =  8  pints  =  4-542  litres. 

i  gallon  distilled  water  =  10  Ibs. 

i  inch  =  2-54  cm. 

i  inch  =  2-54  cm. 

i  foot  =  30-48  cm. 

i  yard  =  91-44  cm. 

i  cm.  =  -39  in. 

i  metre  =  39-37  in- 

i  litre  of  hydrogen  at  o°  C.  and  760  mm.  Hg.  =  0-0896  grams. 


376 


APPENDIX. 


TENSION   OF  AQUEOUS   VAPOUR 
in  millimetres  of  mercury  from  8°  to  20°  C. 


°c. 

mm. 

°C. 

mm. 

°C. 

mm. 

8 

8-0 

M 

11-9 

20 

17-4 

8-5 

8-3 

14-5 

12-3 

20-5 

17-9 

9 

8-6 

15 

12-7 

21 

18-5 

9'5 

8-9 

15-5 

13-1 

21-5 

19-1 

10 

9-2 

16 

13-5 

22 

19-6 

10-5 

9'5 

16-5 

M 

22-5 

2O-2 

ii 

9-8 

i? 

14-4 

23 

20-9 

n-5 

IO-I 

17-5 

14-9 

23-5 

21-5 

12 

10-5 

18 

15*3 

24 

22'2 

12-5 

10-8 

18-5 

r5'8 

24-5 

22-9 

13 

II-2 

19 

16-3 

25 

23-5 

13-5 

"'5 

19-5 

16-8 

INTERNATIONAL    ATOMIC    WEIGHTS. 


Revised 

Revised 

o  =  16 

0  =  10 

Barium 

Ba. 

137-37 

Mercury 

Hg. 

200-6 

Bromine 

Br. 

79-92 

Nitrogen 

N. 

14-01 

Calcium 

Ca. 

40-07 

Oxygen 

0. 

16 

Carbon 

C. 

12-005 

Phosphorus 

P. 

31-04 

Chlorine 

Cl. 

35  "46 

Potassium 

K. 

39-10 

Copper 

Cu. 

63-59 

Silver 

Ag. 

107-88 

Hydrogen 

H. 

i  -008 

Sodium 

Na. 

23. 

Iodine 

I. 

126-92 

Sulphur 

S. 

32-06 

Iron 

Fe. 

55-84 

Tungsten 

W. 

184- 

Lead 

Pb. 

207-2 

Uranium 

TJ. 

238-2 

Magnesium 

Mg. 

24-32 

Zinc 

Zn. 

65-37 

Manganese 

Mn. 

54-93 

APPENDIX. 


377 


SPECIFIC   GRAVITIES    TABLES. 
I.    SULPHURIC  ACID. 


Sp.  Gr. 

Gms.  of 

Sp.  Gr. 

Gms.  of 

I5C 

H2S04 

i£ 

HaSO, 

4° 

in  too  cc. 

4° 

in  100  cc. 

1-840 

175-9 

1-552 

IOO 

1-838 

173-9 

1-542 

98-1 

1-835 

171-7 

1-520 

93-6 

1-833 

170-4 

1-492 

88-95 

1-830 

168-5 

1-420 

74 

1-825 

166-1 

1-380 

66-2 

1-815 

161-8 

1-295 

50 

i  -800 

156-4 

i  -200 

32-8 

II.     HYDROCHLORIC  ACID. 


Sp.  Gr. 

Gms.  of 

Sp.  Gr. 

Gms.  of 

15° 

HC1 

15° 

HC1 

4° 

in  100  cc. 

4° 

in  loo  cc. 

1-160 

36-6 

I-I33 

30 

I-I55 

35-3 

1-113 

25 

1-152 

34'5 

1-091 

20 

1-150 

34>0 

1-056 

12 

I-I45 

32-8 

1-047 

10 

1-140 

3i-5 

1-0375 

8 

378 


APPENDIX. 


III.     SODIUM  AND   POTASSIUM   HYDROXIDES. 


Sp.  Gr. 

Gms.  of 

Gms.  of 

15° 

NaOH 

KOH 

4° 

in  100  cc. 

in  100  cc. 

1-634 

— 

94 

1-615 

— 

90-2 

1-530 

— 

75-6 

i'438 

57'5 

— 

1-397 

50-6 

54-3 

1-370 

46-2 

50-6 

1-332 

40-0 

45-i 

1-190 

20 

25-5 

IV.     AMMONIA. 


Sp.  Gr. 
15° 

Gms. 
NH8 

Sp.  Gr. 
15° 

NH8 
in  100  cc. 

4° 

in  100  cc. 

4° 

•880 

31 

-896 

26-6 

-882 

30-83 

•898 

26-05 

•884 

30-14 

•900 

25'5 

-886 

29-46 

•902 

24-94 

•888 

28-86 

-906 

23-83 

•890 

28-26 

•910 

22-74 

-892 

27-70 

•920 

2O-OI 

-894 

27-15 

•926 

18-42 

APPENDIX. 
V.     ALCOHOL. 


379 


Sp.  Gr. 
I5'560 

Volume 
per  cent. 

Sp.  Gr. 
I5-560 

Volume 
per  cent. 

•79391 

100 

•83065 

9i 

•79891 

99 

•83400 

90 

•80359 

98 

•86395 

80 

•80800 

97 

•87740 

75 

•81217 

96 

•89010 

70 

•81616 

95 

•90214 

65 

•81997 

94 

•91358 

60 

•82365 

93 

•92439 

55 

•82721 

92 

•93445 

50 

BOILING  POINTS. 


Acetic  acid 

119 

Acetone 

56-5 

Alcohol,  anyl     ... 

129-6 

„         butyl,  normal 

117 

„         butyl,  iso 

106 

butyl,  secondary 

99-8 

„         caprylic 

i79'5 

ethyl  

78-3 

„         methyl 

66 

Benzene 

80 

Carbon  bisulphide 

46-2 

Chloroform 

63 

Ether...              

34-18 

Toluol                

in 

380 


APPENDIX. 


Sp.  Gr. 

Gms.  in  100  cc. 

Nitric  acid             

1-42 

99 

Acetic  acid,  "  glacial  "    .  . 

i  -06 

in-i 

Acetic  acid,  "  strong  "    .  . 

1-044 

33 

Sodium  carbonate,  "  saturated  " 

i'i5 

16-2 

STANDARD  ACIDS   AND   ALKALIES. 

A  normal  solution  of  a  substance  contains  in  1000  cc.  that  weight  in  grams, 
which  corresponds  to  i  equivalent  in  grams,  of  available  hydrogen  (1-008 
grams.)  or  its  equivalent. 

Thus  normal  hydrochloric  acid  contains  35-46  +  1-008  =  36-468  grams, 
of  HC1  per  litre. 

Normal  sulphuric  acid  contains 
2-016  +  32-07  +  64 

— - —  -  =  49*043  grams,  of  H2SO4  per  litre. 

It  is  customary  to  employ  normal,  half-normal,  fifth-normal,  etc.,  solu- 
tions according  to  circumstances.  But  it  is  often  much  more  convenient  to 
determine  the  exact  strength  of  a  solution  than  to  adjust  it  to  some  even 
fraction.  For  this  reason  it  is  better  to  express  the  normality  as  a  decimal 
coefficient  rather  than  as  a  fraction.  Thus,  suppose  an  acid  be  found  by 
titration  against  a  known  standard  to  be  0-107  N,  it  can  be  labelled  as  such 
and  used  when  a  solution  about  one-tenth  normal  is  convenient,  the  necessary 
adjustment  in  the  calculation  being  very  simple.  The  relationship  is  not 

N 
so  obvious  if  it  be  labelled  — — ^ . 

In  the  author's  experience  the  simplest  and  most  reliable  starting  point 
for  the  preparation  of  standard  acids  and  alkalies  is  CO2  -  free,  sodium  hydroxide, 
made  and  stored  as  described  on  p.  26.  From  such  a  stock  it  is  a  simple 
matter  to  prepare  acids  or  alkalies  of  any  desired  concentration.  For 
further  details  concerning  the  preparation  and  storage  of  the  alkali  see 
p.  322. 

Thus,  suppose  that  o-i  N.HC1  be  required.  Dilute  pure  concentrated 
hydrochloric  acid  about  90  times  with  distilled  water,  measure  out  25  cc.  and 
titrate  it  with  the  standard  alkali,  using  either  methyl  red  or  phenol  phthalein 
as  the  indicator.  Suppose  that  the  25  cc.  of  dilute  acid  require  13-8  cc.  of 
alkali  which  has  been  found  to  be  0-1965  N.  Then  the  normality  of  the 

13-8 
acid  is  — —   x   0-1965  =  0-1085  N. 

The  acid  can  be  used  as  such,  or  if  exactly  o-i  N.  be  required,  then  8-5  cc. 
of  distilled  water  is  added  to  every  100  cc.  of  the  acid,  thus  bringing  it  to  the 
desired  concentration. 

It  is  important  to  note  that  acids  and  alkalies  act  on  glass,  and  thereby 
suffer  a  change  in  concentration.  This  is  practically  avoided  by  storing  in 
bottles  that  have  been  coated  internally  with  a  fairly  thick  layer  of  paraffin 
wax. 


APPENDIX.  381 


PIPETTES,   ETC. 

Delivery.  In  using  an  ordinary  single-volume  pipette  the  fluid  is  drawn 
by  suction  just  above  the  mark  and  closed  with  the  finger.  The  lower  end 
of  the  pipette  is  then  allowed  to  touch  the  side  wall  of  the  bottle 
or  beaker  and  the  fluid  run  out  till  the  meniscus  is  exactly  at 
the  mark,  the  eye  being  level  with  the  meniscus.  The  fluid 
is  then  allowed  to  run  out  into  the  desired  vessel  and  is  then 
drained  for  15  seconds  with  its  point  touching  the  wall  of  the 
vessel.  The  majority  of  pipettes  are  calibrated  for  such  a 
delivery,  but  for  certain  operations  the  author  prefers  to  use 
pipettes  which  are  calibrated  in  such  a  way  that  they  have  to 
be  blown  out  after  draining  as  above  for  15  sees.  The  reason 
why  these  are  sometimes  preferable  is  that  the  amount  left  in 
the  nozzle  of  the  pipette  after  drainage  may  alter  considerably 
with  variations  in  the  surface  tension  of  the  fluid  measured. 
It  is  suggested  that  such  pipettes  calibrated  for  delivery  by 
blowing  should  be  engraved  with  the  letter  "B"  to  distin- 
guish them  from  pipettes  calibrated  for  drainage  "  D." 

Ostwald  pipettes  (see  fig.  48)  are  always  calibrated  for 
delivery  by  being  completely  blown  out.  The  orifice  must 
be  so  narrow  that  it  takes  about  30  seconds  for  the  delivery 
of  i  cc.  They  are  filled  by  suction  to  above  the  mark  and 
then  closed  with  the  finger.  The  exterior  is  wiped  with  a 
piece  of  filter  paper  and  the  fluid  run  to  the  mark  by  holding 
the  point  on  the  filter  paper.  The  fluid  is  then  allowed  to  fall 
out  by  its  own  weight,  the  delivery  being  completed  by  blowing 
Fig.  48.  out  whilst  drawing  the  point  of  the  pipette  up  the  sides  of  the 
Ostwald  receiving  vessel. 

pipette.  Burettes.     The    chief  precautions  to  be  taken  are  to  allow 

time  for  proper  drainage,  and  to  be  sure  that  the  meniscus 
is  read  in  the  same  way  at  every  operation.  The  author  prefers  to  use 
burettes  that  have  the  marks  engraved  as  complete  circles,  and  to 
read  the  meniscus  by  means  of  a  piece  of  black  paper  held  behind 
the  burette.  The  black  paper  is  pasted  on  to  a  piece  of  white  card, 
which  is  sharply  folded  at  the  black  edge  (see  fig.  49).  The  black  edge 
is  held  against  the  back  of  the  burette  a  trifle  below  the  meniscus,  the 
slanting  white  card  reflecting  the  light.  The  meniscus  appears  as  a  very  sharp 
black  line.  In  order  to  avoid  parallax  it  is  important  that  the  eye  should  be 
exactly  at  the  level  of  the  meniscus.  To  ensure  this  for  very  accurate  work 
the  author  has  constructed  the  device  shewn  in  fig.  50.  Should  the  fluid  be 
run  rapidly  out  of  a  burette  ample  time  must  be  allowed  for  proper  drainage, 
the  meniscus  gradually  moving  upwards  as  the  fluid  runs  down  the  side  of  the 
burette.  Details  of  the  methods  employed  for  the  calibration  of  pipettes  and 
burettes  will  be  found  in  most  standard  works  on  Quantitative  Chemical 
Analysis.*  It  is  essential  that  pipettes  and  burettes  should  be  clean  and 
free  from  grease.  Very  considerable  errors  can  be  caused  by  variations  in 

*  Representative  Procedures  in  Quantitative  Chemical  Analysis,  by  F   A. 
Gooch  (Chapman  &  Hall,  London,  1916),  can  be  recommended. 


382 


APPENDIX. 


the  amount  of  fluid  adhering  in  the  form  of  drops  in  a  greasy  pipette  or 
burette.  Should  the  burette  have  a  glass  stopcock  this  must  be  greased. 
Under  these  circumstances  the  fluid  must  always  be  run  out  of  the  burette 
by  the  stopcock.  If  it  is  emptied  by  opening  the  tap  and  inverting,  the  inner 
wall  of  the  vessel  is  almost  certain  to  become  greasy.  When  this  occurs, 


Fig.  49.  Fig-  50. 

Author's  device  for  reading  burettes. 

A  is  a  draw  tube  containing  a  lens  B.  E  is  a  paper  disc 
pierced  with  a  small  hole.  The  tube  C  and  D  are  blackened. 
The  whole  is  fastened  to  a  wooden  block,  F.  This  is  firmly 
held  to  the  burette  by  a  clip  and  spring.  By  placing  the 
finger  in  the  groove  G  and  pressing  with  the  thumb  on  H,  the 
tube  can  be  moved  up  and  down  until  the  meniscus  is 
sighted.  L  is  a  piece  of  paper,  the  lower  half  of  which  is 
blackened.  The  device  is  for  reading  to  one-tenth  of  the 
ordinary  graduations  of  the  burette.  The  nearest  tenth  is  best 
obtained  by  the  method  described  above. 


wash  the  burette  out  with  water.  Fill  it  with  strong  (40%)  soda.  Run  this 
out  and  then  wash  it  out  repeatedly  with  tap  water.  Now  fill  the  burette 
with  chromic  acid  cleaning  fluid  and  allow  it  to  stand  over-night.  Wash  out 
as  before.  The  burette  will  now  keep  free  from  grease  for  some  time  if  properly 
used. 


APPENDIX. 


383 


FOLIN'S    FUME-ABSORBER. 

Since  the  fumes  arising  from  the  incineration  of  a  substance  with  boiling 
sulphuric  acid  are  extremely  irritating,  that  operation  should  be  conducted  in 
a  fume  chamber  or  under  a  hood.  But  it  is  preferable  to  use  the  very  con- 
venient apparatus  devised  by  Folin,  since  the  removal  of  the  condensation 
water  materially  accelerates  the  incineration. 


Fig.  51.     Folin's  fume-absorber. 

The  apparatus  consists  of  a  bulb  C  (i£  inches  in  diameter)  blown  into  a 
piece  of  fths.  tubing.  The  lower  end  has  blown  into  it  an  open  piece  of 
narrow  tubing  2^  inches  in  length.  The  bulb  rests  on  the  neck  of  the  flask  or 
test-tube  in  which  the  incineration  is  conducted.  To  the  upper  end  of  the 
tube  is  fixed  a  piece  of  narrow  glass  tubing  which  is  bent  at  a  convenient  angle 
and  connected  by  a  short  length  of  rubber  tubing  to  a  glass  tube  B.  This  is  of 
such  a  size  that  it  just  slips  into  one  limb  (A)  of  a  T-piece.  This  is  fastened  to 
a  board  or  shelf  by  metal  clips.  One  end  of  the  horizontal  limb  of  the  T-piece 
is  connected  to  a  suction  pump,  the  other  end  being  joined  by  a  piece  of  pressure 
tubing  (D)  and  a  length  of  metal  tubing  (E)  to  another  T-piece.  This  can  be 
connected  to  another  fume-absorber.  One  good  pump  is  sufficient  for  3 
absorbers.  Those  not  in  use  should  be  stoppered  with  corks.  It  is  sometimes 
necessary  to  fit  a  rubber  collar  on  to  B,  so  that  good  suction  is  obtained  through 
C.  Owing  to  the  rubber  joints  the  angles  of  the  limbs  A  and  F  can  be  varied 
to  suit  the  heights  of  the  vessels  in  which  the  incineration  is  being  conducted. 

The  fumes  are  carried  over  by  the  air  current  into  the  pump,  a  wash  bottle 
containing  soda  being  interposed  to  prevent  damage.  The  condensation 
water  collects  in  the  pocket  below  C  and  can  be  removed  by  inverting  the 
fume-absorber  at  the  end  of  the  operation. 

By  inverting  a  funnel  over  an  evaporating  basin,  and  arranging  the 
apparatus  so  that  the  end  of  the  funnel  fits  loosely  into  the  neck  of  the  absorber, 
the  fumes  from  boiling  nitric  acid  can  be  carried  off. 


APPENDIX. 


COLORIMETERS. 

A  high  grade  colorimeter  is  a  necessary  adjunct  of  a  Biochemical  Labora- 
tory, a  number  of  important  analyses  being  made  by  its  use. 


Fig.  52.     Duboscq's  Colorimeter. 
Inset  shows  construction  of  vernier  scale. 

The  best  known  instrument  is  that  of  Duboscq,  which  is  shewn  in  fig.  52. 
The  standard  solution  is  placed  in  one  of  the  cups,  B,  and  the  unknown  solution 
in  the  other  cup.  The  plungers,  D,  are  either  cylindrical  hollow  cups,  closed 
at  the  bottom,  or,  preferably,  are  made  of  a  solid  piece  of  optically  clear  glass. 
They  can  be  moved  up  and  down  by  turning  the  screw  E,  which  works  on  a 
rack  and  pinion.  The  height  of  the  bottom  of  the  plungers  from  the  bottom 
of  the  cups,  that  is  the  depth  of  the  solution  used,  can  be  read  by  means  of  a 
scale  and  vernier  at  the  back  of  the  instrument.  The  standard  is  set  at  a  given 
height  (say  15  mm.)  and  the  height  of  the  other  plunger  adjusted  until  exact 
equality  of  tint  is  obtained.  The  light  is  reflected  through  the  solutions  from 
A,  which  is  either  a  mirror  or  a  piece  of  opal  glass.  After  passing  through 
the  layers  of  the  fluids  on  the  two  sides  the  light  falls  on  to  the  prisms  shewn 
in  K  of  fig.  53.  These  prisms  are  contained  in  the  case  marked  J  in  fig.  52. 
The  light  then  passes  through  the  eye  piece  as  shewn. 


APPENDIX. 


385 


There  are  several  important  points  of  detail  that  must  be  attended  to 
before  accurate  results  can  be  obtained. 

(1)  See  that  the  zero  points  of  the  scales  -... 
are  correct,  by  carefully  lowering  the  plungers                         o  *& 
until    they  touch    the    cups    and    noting    the 

readings. 

(2)  See  that  the  prisms  and  eye  piece  are 
clean.     Specks  of  dust  seen  in  the  field  are  apt 
to  lead  to  erroneous  judgments.      The  prisms 
can  be  removed  and  carefully  cleaned  with  a 
pointed  match  covered  with  two  or  three  layers 
of  silk.     Great   care   must  be  taken  to  avoid 
breaking  the  prisms  away  from  the  cement. 

(3)  See  that  the   illumination  of  the  two 
fields  is  equal.      This  is  b,est  tested  by  placing 
a    coloured    solution    (such  as   a    2-5  per  cent, 
solution  of  potassium  dichromate)  in  both  cups, 
placing  one  plunger  at  15  mm.  and  adjusting  the 
other    until   the   two   fields   have   an   identical 
appearance.      The    other    plunger   should    also 
be  at  15  mm.     Attention  must  be  paid  to  the 
point  considered  below. 

(4)  Folin    has    suggested    that    the    best 
place   for  an  instrument  is  in  the    middle   of 
the  Laboratory,  so  that  the  eye  is  not  dazzled 
by  the  light  from  the  window.     Retinal  fatigue 
will  undoubtedly  cause  very  serious  errors  and 
inconsistencies,   and  Folin's  recommendation  is 
valuable. 

(5)  A    comfortable   body  position   is   im- 
portant.    For  some  reason  the  best  results  are 
obtained  when  the  observer  is  in  an  unstrained 
position.     Folin  suggests  that  the  best  way  of 
using  the  apparatus  is  to  place  it  on  a  stool  about 
the  same  height  as  an  ordinary  chair,  and  to  sit 
at  the  side  of  the  instrument.     His  method  of 
reading  the  unknown  is  to  place  the  standard 
solution  into  both  cups  and  to  set  them  at  the 
same  height.     The  instrument  being  adjusted, 
both  fields  should  look  alike,  and  the  eye  gets 
accustomed  to  the  appearance.     The  standard 
in  one  cup  is  replaced  by  the  unknown,   and 
one  very  careful  observation  is  taken.      When 
making  a  series  of  comparisons,  he  re-reads  the 
standard  against  itself  after  each  of  two   un- 
knowns. 

Kober's  Colorimeter*  is  a  great  advance  on  Duboscq's.  The  manu- 
facturers (Klett  Manufacturing  Co.,  New  York,  U.S.A.)  kindly  sent  one  to 
Cambridge  for  trial.  It  has  been  found  admirable  in  every  way,  and  is  always 
used  now  in  preference  to  the  various  patterns  of  Duboscq's.  Kober's 
instrument  can  also  be  used  as  a  Nephelometer,  i.e.  for  estimating  substances 


Fig.  53.  Diagram  of  path 
of  rays  in  Duboscq's 
Colorimeter.  Below 
are  representations  of 
the  appearance  of  the 
field  under  different 
conditions,  that  on  the 
left  with  no  fluid  in  B, 
and  that  on  the  right 
when  the  tints  are 
matched. 


*  Journ.  of  Biol.  Chem.,  xxix.,  p.  155. 


BB 


386 


APPENDIX. 


jmiiimiimiiiniiiiiiniiiiuimi 


Fig.  54.     Kober's  Colorimeter.    Inset  shews  the  form 
of  two  of  the  cups. 


APPENDIX.  387 

by  the  density  of  a  cloudy  precipitate.  Though  this  book  does  not  contain  an 
example  of  this  method  of  analysis,  it  is  of  growing  importance,  being  valuable 
when  only  very  small  amounts  of  material  are  available.  It  is  also  supplied, 
if  desired,  with  an  excellent  lamp  house,  so  that  it  can  be  used  at  night.  In 
fact,  it  is  more  satisfactory  to  use  artificial  light,  since  by  means  of  mirrors 
the  illumination  of  the  two  fields  can  be  made  exactly  equal. 

The  instrument  is  shewn  in  fig.  54,  and  it  will  be  seen  that  the  plungers 
are  fixed  and  the  cups  movable.  This  does  away  with  the  space  between  the  top 
of  the  cup  and  the  prism,  which  is  apt  to  allow  an  indefinite  amount  of  light 
through  in  a  Duboscq.  The  prisms  are  enclosed  and  so  remain  free  from  dust. 
The  use  of  dark  glass  for  the  sides  of  the  cups  and  the  plungers,  the  absence 
of  cements,  improved  mechanical  arrangements  for  adjusting  the  zero  and 
moving  the  cups,  and  the  reduction  in  the  volume  of  fluid  necessary,  all 
combine  to  make  the  instrument  nearer  perfection  than  anything  yet 
introduced.* 

The  calculations  necessary  in  colorimetric  work  are  very  simple.  The 
assumption  is  that  the  depth  of  colour  is  proportional  to  the  concentrations 
of  the  substance  in  the  standard  and  in  the  unknown.  Also  that  the  depth 
of  field  of  the  solutions  that  must  be  taken  to  get  equality  of  tint  vary  in- 
versely as  the  intensity  of  the  colour  produced,  and  therefore  as  the  concen- 
trations in  the  standard  and  unknown.  So  if  the  standard  contains  a  certain 
amount  of  material  in  a  given  volume  and  the  unknown  is  made  up  to  the  same 
volume,  then 

Amount  in  standard       Reading  of  unknown 
Amount  in  unknown  ~~  Reading  of  standard 


*  Both  instruments  can  be  obtained  from  Messrs.  Baird  and  Tatlock 
(London). 


388 


APPENDIX. 


TORSION   BALANCE. 

This  instrument  is  of  value  for  the  rapid  weighing  of  small  amounts  of 
substances,  such  as  blood  taken  from  a  finger-prick,  etc. 

The  instrument  is  used  as  follows  for  the  weighing  of  blood  on  a  piece  of 
absorbing  paper,  as  for  the  micro-analysis  of  sugar  (see  p.  254). 


Fig-  55 

Remove  the  clip  G  and  paper  from  the  arm  D.  Move  C  until  the  indicator 
A  is  at  zero  on  the  scale.  See  that  the  lever  E  is  in  such  a  position  that  F 
points  to  "  Free."  The  movable  arm  B  should  now  be  at  O.  If  this  is  not 
so,  bring  B  to  O  by  means  of  an  adjusting  screw  on  the  back  of  the  instrument. 

Now  set  F  to  "  Stop  "  by  means  of  E.  Hang  the  clip  and  paper  on  to  D, 
seeing  that  the  paper  hangs  freely.  By  means  of  C  move  the  lever  A  to  mark 
about  120  mgm.,  set  F  to  "  Free,"  and  then  move  C  until  B  is  at  O.  The 
reading  at  A  is  the  weight  of  the  paper  and  the  clip.  After  the  blood  has  been 
drawn  on  to  the  paper,  the  weight  is  again  taken  as  before.  This  should  be 
done  as  rapidly  as  possible  to  avoid  errors  due  to  evaporation. 

The  paper  and  clip  should  never  be  put  on  or  taken  off  D  with  F  at 
"  Free."  The  indicator  should  be  set  at  the  approximate  weight  before  the 
spring  is  released.  By  taking  these  precautions  the  instrument  will  remain 
reliable  for  a  very  long  time. 


APPENDIX. 


389 


THE   PREPARATION   OP   CERTAIN  REAGENTS  AND 
LABORATORY  REQUISITES. 

Acid  potassium  phosphate,  see  p.  24. 
Acid  potassium  phthalate,  see  p.  24. 

Almtn's  reagent.  4  grams,  of  tannic  acid  in  8  cc.  of  strong  (33  per  cent.) 
acetic  acid  and  190  cc.  of  50  per  cent,  alcohol. 

Alpha-naphthoL     i   per  cent,  in  strong  alcohol. 

A  mmonium  molybdate.  Rub  up  75  grams,  of  crystalline  ammonium  molyb- 
date  with  300  cc.  of  strong  ammonia.  When  it  has  dissolved  gradually  add 
the  solution  to  a  mixture  of  900  cc.  of  concentrated  nitric  acid  (sp.  gr.  1-42) 
and  400  cc.  of  distilled  water,  cooling  thoroughly  during  the  addition.  Add 
1600  cc.  of  distilled  water  and  filter,  if  necessary. 

Ammonium  oxalate,   0-2  N.     1-42   per  cent,  of   (COO.NH4)2H2O. 

A  mmonium  sulphate,  saturated  solution.  Boil  800  grams,  of  pure  crystal- 
line (NH4)2SO4  with  about  i  litre  of  distilled  water.  Filter  when  cold. 

A  mmonium  sulphide  is  usually  purchased.  Can  be  prepared  by  saturating 
ammonia  (i  part  of  concentrated  to  2  parts  of  water)  with  sulphuretted 
hydrogen  and  then  adding  to  this  one-third  of  its  volume  of  ammonia  of 
the  same  dilution. 

Asbestos  pulp  for  Gooch  crucibles,  etc.  Cut  some  long-fibred,  soft 
asbestos  into  pieces  about  a  quarter  of  an  inch  long  and  digest  with  concen- 
trated hydrochloric  acid  in  a  large  flask  in  a  water  bath  for  an  hour,  shaking 
thoroughly  at  intervals.  Filter  through  a  plate  or  on  a  Buchner  under  light 
suction  and  wash  thoroughly  with  water.  Transfer  to  a  large  wide-neck 
stoppered  bottle  and  shake  thoroughly  with  water.  A  good  quality  asbestos 
forms  a  sludge,  which  allows  of  rapid  filtration,  provided  that  it  be  not  unduly 
compressed  by  too  great  a  suction. 

To  prepare  a  Gooch  crucible  for  gravimetric  analysis,  set  up  the  apparatus 
shewn  on  p.  279.  Pour  on  enough  of  the  asbestos  sludge  to  form  a  layer 
i  to  2  mm.  thick.  On  this  place  a  perforated  porcelain  plate,  the  diameter 
of  which  is  slightly  less  than  that  of  the  crucible,  and  then  add  another  layer 
of  asbestos.  Filter  under  light  pressure  and  pass  water  through  until  the 
filtrate  is  absolutely  clear.  The  crucible  can  then  be  dried  in  a  hot  air  oven 
at  1 10°  C.,  cooled  and  weighed.  The  drying  and  weighing  should  be  repeated 
until  the  weight  is  constant.  It  should  then  be  tested  by  passing  about  500  cc. 
of  water  through  it,  drying  and  weighing.  If  constant  it  is  ready  for  use. 
The  same  crucible  can  be  used  for  a  large  number  of  determinations. 

Barfoed's  reagent,  see  p.  108. 

Barium  chloride,  N.     122  grams,  of  BaCLj^HgO  to  i  litre. 

Baryta  mixture.  One  vol.  of  barium  chloride  solution  is  added  two  vols. 
of  baryta  water. 

Baryta  water.  One  part  of  crystalline  barium  hydroxide  is  dissolved  in 
15  parts  of  boiling  water  and  filtered  hot.  The  filtrate  on  cooling  throws 
down  crystals  of  barium  hydroxide.  The  supernatant  fluid  is  baryta  water.  It 
is  about  0-25  Normal. 

Benedict's  solution,  qualitative,  seep.  107. 
Benedict's  solution,  quantitative,  see  p.  127. 


39°  APPENDIX. 

Benzidine  hydrochloride,  see  p.  357. 

Bromine  water,  saturated.  Made  by  shaking  bromine  with  cold  distilled 
water. 

Brucke's  reagent,  see  p.  37. 

Calcium  chloride,  normal.  55-5  grams,  pure  anyhdrous  CaCl2,  dissolved  in 
water,  made  up  to  i  litre  and  filtered.  One-fifth  normal  is  a  convenient 
strength  for  certain  exercises. 

Charcoal,  adsorbent.  The  author  has  found  that  certain  samples  of  the 
charcoal  prepared  by  the  Chemical  Warfare  Department  for  filling  protective 
gasmasks  are  highly  efficient,  being  superior  to  Merck's  "blood  charcoal." 
It  is  hoped  that  carefully  selected  supplies  will  shortly  be  obtainable  from 
Messrs.  Baird  and  Tatlock's  and  other  dealers. 

Chromic  acid  cleaning  fluid.  Ten  per  cent,  of  chromic  acid  in  water,  or 
10  per  cent,  of  potassium  bichromate  dissolved  in  10  percent,  (by  volume) 
of  sulphuric  acid. 

Cochineal  tincture,   see  p.   354. 
Collodion  solution,  see  p.  3. 

Congo  red  paper.  White  filter  paper  is  thoroughly  wetted  with  a  0-2  per 
cent,  solution  of  congo  red  in  water.  The  paper  is  pinned  up  till  dry  and  cut 
into  strips.  It  is  turned  blue  by  strong  acids. 

Copper  sulphate.  200  grams,  of  pure  crystalline  CuSO4.5H2O  are  dissolved 
in  distilled  water  by  the  aid  of  heat,  cooled  and  made  up  to  i  litre.  For  the 
biuret  reaction  a  i  per  cent,  solution  is  prepared  by  diluting  5  cc.  to  100  cc. 

Ehrlich's  reagent  for  indol.    Para-dimethyl-amido-benzaldehyde  4  parts 
Alcohol  (95  to  98  per  cent.)      . .      380       ,, 
Concentrated  hydrochloric  acid         80       ,, 

Esbach's  reagent,  see  p.  36. 
Fehling's  solution,  see  p.  106. 
Ferric  chloride,  10  per  cent. 
Glyoxylic  reagent,  see  p.  39. 

Grease  paint,  for  marking  beakers,  etc.  Dilute  Brunswick  Black  to  the 
desired  consistency  with  naphtha  or  benzene.  Apply  with  a  fine  brush.  Can 
be  scraped  off  or  removed  by  means  of  a  pad  of  cotton  wool  soaked  in  naphtha. 

Gunzberg's  reagent.  Dissolve  2  grams,  phloroglucin  and  i  gram,  of 
vanillin  in  30  cc.  of  absolute  alcohol.  The  solution  should  be  freshly 
prepared,  but  it  can  be  preserved  for  a  certain  time  in  dark  bottles. 

Ink  for  writing  on  glass. 

A.  13  per  cent,  solution  of  shellac  in  strong  alcohol. 

B.  13  per  cent,  solution  of  borax  in  distilled  water. 

Mix  3  parts  of  A  with  5  parts  of  B,  and  add  some  stain  in  alcoholic  or  aqueous 
solution. 

Iodine  solution.  About  o-i  N.  Dissolve  25  grams,  of  potassium  iodide  in 
about  200  cc.  of  distilled  water  in  a  stoppered  flask.  Add  12-7  grams,  of  iodine 
and  shake  till  dissolved.  Make  up  to  i  litre  with  distilled  water.  For  many 
purposes  this  can  be  diluted  10  times  or  even  more  with  distilled  water,  but 
these  weak  solutions  should  be  prepared  as  required. 

Lead  acetate  (basic).  Boil  464  grams,  of  normal  lead  acetate  and  264 
grams,  of  litharge  in  1500  cc.  of  distilled  water  for  half  an  hour  with  constant 
stirring.  Cool  and  filter.  Or  use  a  saturated  solution  of  the  commercial 
basic  lead  acetate. 


APPENDIX.  391 

Lead   acetate    (normal).     Saturated  solution. 

Litmus  solution.  Extract  the  crushed  litmus  several  times  with  warm 
distilled  water,  mix  the  extracts  and  filter.  Adjust  the  solution  to  a  neutral 
tint  by  means  of  hydrochloric  acid.  The  sensitiveness  of  the  indicator  is 
much  increased  by  dialysing  it  against  distilled  water.  A  drop  or  two  of 
chloroform  may  be  added  to  the  solution  to  prevent  the  growth  of  organisms. 

Mercuric  chloride.     Saturated  solution,  about  8  per  cent. 

Mercuric  nitrate.  A.  To  160  cc.  of  concentrated  nitric  acid  (sp.  gr.  1-42) 
in  a  beaker  add,  in  small  portions,  220  grams,  of  red  mercuric  oxide.  Stir  well 
and  then  add  160  cc.  of  distilled  water.  Heat  till  the  oxide  has  dissolved. 
Cool  and  nearly  neutralise  by  adding  75  cc.  of  N.  soda.  Make  up  to  i  litre 
and  filter.  Preserve  in  a  dark-coloured  bottle.  This  solution  is  used  for 
removing  various  nitrogenous  substances  from  urine  when  estimating  small 
quantities  of  sugar  (see  p.  345). 

B.  To  143  grams,  of  pure  mercury  in  an  evaporating  basin  add  200  cc.  of 
concentrated  nitric  acid  (sp.  gr.  1-42).  Heat  until  thick  fumes  are  evolved 
and  then  turn  out  the  gas.  When  the  reaction  has  ceased  light  the  flame 
again  and  evaporate  down  to  about  80  cc.  Gradually  add  about  1500  cc. 
of  water.  Cool  and  make  up  to  2  litres. 

Mercuric  sulphate  reagent  for  tryptophane.      See  p.  89. 

Millon's  reagent.     See  p.  39.     It  is  usually  purchased. 

Nessler's  solution.     Folin  and  Denis,  Journ  Biol.  Chem.,  xxvi.,  p.  478. 

Phosphotungstic  acid.     Two  per  cent,  in  5  per  cent,  sulphuric  acid. 

Picric  acid,  saturated  solution,  about  1-2  per  cent. 

Potassium  ferricyanide.  Saturated  solution,  prepared  by  grinding  the 
solid  with  cold  water  in  a  mortar. 

Potassium  jerrocyanide,  5  per  cent. 
Roberts'  reagent,  see  p.  304. 

Sodium  hypobromite.  Dissolve  100  grams,  of  caustic  soda  in  250  cc.  of 
water.  Cool.  Cautiously  add  25  cc.  of  bromine,  cooling  thoroughly  at 
intervals.  It  must  be  recently  prepared. 

Soluble  starch.  250  grams,  of  potato  starch  is  placed  in  a  litre  flask.  It  is 
treated  with  a  mixture  of  375  cc.  of  water  and  125  cc.  of  pure  concentrated 
hydrochloric  acid  and  the  mixture  thoroughly  shaken  until  the  whole  of  the 
starch  has  been  wetted  by  the  acid.  It  is  allowed  to  digest  at  room  tempera- 
ture for  8  days,  being  frequently  shaken.  The  acid  is  then  poured  off,  the 
residue  repeatedly  washed  with  distilled  water  and  then  filtered  on  a  Buchner. 
To  remove  the  last  traces  of  acid,  which  inhibit  the  action  of  enzymes,  it  is 
advisable  to  suspend  the  starch  in  a  buffer  solution  of  PH  =  7.  This  can  be 
prepared  approximately  by  treating  50  cc.  of  0-2  M.  acid  potassium  phosphate 
(see  p.  24)  with  30  cc.  of  0-2  N.  soda  and  diluting  to  500  cc.  After  standing 
for  some  time  with  frequent  shakings  the  starch  is  again  washed  by  decanta- 
tion  with  distilled  water,  filtered  on  a  Buchner  and  dried  in  the  air.  Solutions 
are  prepared  in  the  manner  described  for  starch  paste  (p.  120). 

Stokes'  reagent,  see  p.  245. 
Sulphosalicylic  acid,  see  p.  37. 
Tannic  acid,  see  Almen's  reagent. 

Tap  grease.  Heat  together  in  a  crucible  on  a  sand  bath  i  part  of  soft 
rubber  (free  from  inorganic  filling  matter),  i  part  of  paraffin  wax  and  2  to  3 
parts  of  vaseline.  Stir  thoroughly  until  the  rubber  has  completely  dissolved. 


INDEX. 


Absorption  spectra,  243 

chart  of,  372 
Acetate  buffers,  28 
Acetic  acid,    dissociation   constant 

of,  12 
Aceto-acetic  acid,  314 

estimation  of,  347 

preparation  of,  316 

removal  of,  340 

tests  for,  316 
Acetone, 

estimation  of,  347 

tests  for,  316 

Achromic  point  method,  188,  190 
Acid,  excretion  of,  274 
Acidity,  14 

estimation  of,  275 

of  gastric  juice,   196 

of  urine,  273 
Acid  haematin,  247 
Acid  haematoporphyrin,  248 
Acidosis,  275,  315 
Acid  phosphates,  283 
Acid,  standard,  27,  380 
Acrolein,  160 

Active  hydrochloric  acid,  196 
Adenine,  62,  298 
Adjusting  reaction,  277 
Alanine,  68 
Albumins,  45 

boiling  test  for,  303 

crystallisation  of,  50 

detection  of,  363 

heat  coagulation,  42 

in  milk,  170 

in  urine,  302 

preparation  of,  47 

properties  of,  45 

removal  of,  48 

serum,  47 

tests  for,  47 
Albuminoids,  34.  58 
Albuminuria,  302 
Albumoses  (see  proteoses),  52 

detection  of,  365 

formation  of,  53 

hetero,  53 

in  urine,  304 


primary,  53 

secondary,  54 

separation  of,  53 
Alcohol, 

specific  gravity  of,  379 
Aid  oses,  40 
Alkalies,  standard,  26 
Alkaline  haematin,  247 
Alkaline  haematoporphyrin,  248 
Alkaline  tide,  273 
Alkali  reserve,  275 
Alkaloidal  reagents,  36 
Allan toin,  292 
Alloxan,  292,  295 
Alloxantin,  295 
Alpha-napthol  test,   in,   112 
Amines,  223 
Amino-acids,  67 

estimation  of,  214,  333 

in  urine,  333 

in  various  proteins,  71 

methods  of  separation,  70 

reactions  of,  69 
Ammonia, 

estimation  of,  329 

in  urine,  274 

specific  gravity  of,  378 
Ampholytes,  31 
Amphoteric  electrolytes,   31 
Amylase,  117,  186 
Amylopectin,  117 
Amylopsin,  220 
Analysis  of 

blood,  251  et  seq. 

fluids,  361 

gastric  juice,  195  et  seq. 

solids,  370 

urine,  321  et  seq. 
Anti-ferments,  186 
Aqueous  vapour, 

tension  of,  376 
Arabinose,   101 
Arginine,  69 
Asbestos  filters,  389 
Asparagine,  81 
Aspartic  acid,  81 
Asymmetric, 

carbon  atom,   147 


INDEX. 


393 


Asymmetric  compounds, 

resolution  of,  151 
Atomic  weights,  376 
Autolysis,  228 

Bacteria, 

nutrient  media  for,  226 
Bacterial  decomposition,  222 
Bang's  method  for 

chlorides,  257 

glucose  in  blood,  253 
Barfoed's  test,   108,  114,  115 
Beckmann's  method,  8 
Bence-Jones'  protein,  304 
Benedict's  method 

for  creatine,  339 

for  sugar,  127 

for  sugar  in  blood,  251 

for  sugar  in  urine,  345 

for  sulphur,  356 
Benedict's  sulphur  reagent,  356 
Benzidine 

method  for  sulphates,  357 

test  for  blood,  306 
Bertrand's  method  for  sugar,  126 
0-iminazol-ethylamine,  223 
Bial's  test,  312 
Bile,  264 
Bile  pigments,  267 

in  urine,  307 
Bile  salts,  265 

action  on  lipase,   158 

in  urine,  308 
Bilirubin,  267 
Biliverdin,  267 
Biuret,  41,  287,  291 
Biuret  reaction  for  proteins,  40 
Blood, 

chlorides,  257 

coagulation  of,  234 

detection  of,  305 

glucose  in,  249 

haemolysis  of,  239 

in  urine,  305 

laking  of,  239 

non-protein  nitrogen  of,  260 

plasma,  234 

serum,  234 
Boiling  points,  379 
Bread,  173 
Bromine  reaction 

for  trytophane,  94,  217 
Briicke's  reagent,  37 
"Buffers,"   17 
Buffer  solutions, 

standard,  27 
Burettes,  381 


Cadaverine,  225 
Calcined  milk,  206,  213 
Calcium  phosphates 

in  milk,  172 

in  urinary  sediments,  319 

in  urine,  283 
Calcium  salts 

in  clotting  of  blood,  234 

in  clotting  of  milk,  208 

in  heat  coagulation  of  proteins, 

43.  45 

in  milk,  172 

in  urine,  280,  284 
Cane  sugar,  116 

estimation  of,  142 

test  tor,  117 
Caramel,  105 
Carbamide,  see  urea 
Carbohydrates,  100 

detection  of,  365 

estimation  of,   125 

in  proteins,  34,  57 
Carboxy-haemoglobin,  295 
Carmine  fibrin,  204 
Casein,  166,  167 

action  of  rennin  on,   167,  207 

digestion  of,  87,  213 

iso-electric  point,  n 

solution  for  enzyme  work,  213 
Caseinogen,  34 
Cerebrosides,  165 
Charcoal,  adsorbent,  352 
Cheese,  1 72 
Chlorides, 

detection  of,  284 

estimation  of,  199,  257,  352 

in  blood,  257 

in  urine,  281,  352 
Cholesterol,  161,  239,  268 

preparation  of,  162 

reactions  of,  162 
Choline,   164,  165 
Chromic  period,  191 
Chromoproteins,  34 
Claisen  flasks,  99 
Cleaning  fluid  for  glass,  390 
Clotting, 

see  coagulation 
Coagulation 

of  blood,  234 

of  milk,  207 

of  proteins,  by  alcohol,  37 

of  proteins,  by  heat,  42 
Co-ferments,   185 
Cole -and  Onslow 

comparator,  21,  276 

on  nutrient  media,  226 


394 


INDEX. 


Cole's  apparatus  for 
automatic  delivery,   191 
reading  burettes,  382 
standard  heating  power,  136 
Cole's  method  for 
acidity  of  urine,  275 
ammo-acids,   215 
diastase,  193 
formol  titration,  333 
lactose,  139 
micro-Kjeldahl,  261 
total  nitrogen,  327,  329 
uric  acid,  341 
Cole's  test  for 

bile  pigments,  267,  307 
glucose,   109 
lactose  in  urine,  313 
sugar  in  urine,  310 
Collagen,   58 
Collodion  solution,  3 
Colloids,   i 

electrical  properties  of,  9 
precipitation  of,    10,    u,    13 
Colorimeters, 
Duboscq's,  384 
Kober's,  385 
Colorimetric 

determination  of  PH,  29 
Colour  reactions  of  proteins,  38 
Comparators, 
large,  276 
small,  20 
Congo  red,  22 
papers,  390 
Copper 

ammoniacal,  preparation  of,  291 
Copper  salt, 

of  aspartic  acid,  82 
of  glycine,  77 
Creatine,  178 

conversion  into  creatinine,  179 
estimation  of,  338,  339 
from  meat  extract,   178 
in  urine,  298 
Creatinine,  178,  298 
estimation  of,  336 
Jaffe's  test,  179,  300 
origin  ot,  298 
preparation  of,  299 
Wehl's  test,  179,  301 
Zinc  chloride  compound,  300 
Cresol,  223,  282 
Cryoscopy,  5,  272 
Cyanuric  acid,  287,  291 
Cysteine,  41 
Cystine,  61 

in  proteins,  41 


in  urine,  319 
preparation  of,  82 
properties  of,  83,  84 
Cytosine^  63 

Deposits  in  urine,  319 
Dextrins,   118 

detection  of,  366 

distinction  from  glycogen,  121 

formation  of,  118,  121 

malto,  119 

reactions  of,   121 

stable,  118 
Dextrose,  101 

see  glucose 
Diabetes,  250,  308 
Dialysed  iron,   10 
Dialysing  membranes,  2 
Dialysis,  2,  3 

of  serum,  48 
Diastase,  urinary,  359 
Diffusion,  2 
Digestion, 

of  carbohydrates,   187,  220 

of  fats,  157 

of  nucleo-proteins,  60 

of  proteins  by  erepsin,  218 

of  proteins  by  pepsin,  53,  201 

of  proteins  by  trypsin,  212 
Dilution,  effect  of,  on  reaction,  1 
Disaccharides,  113,  221 
Dissociation  constant,  18 
Distillation 

in  vacuo,  73,  99 
Distributor,  191 
Dreyer's 

dropping  pipette,  24 
Dubos.q's  colorimeter,  384 
Dulcite,  104 
Dunstan's  test 

for  glycerol,  160 

Earthy  phosphates,  283,  363 
Edestin  method  for  pepsin,  204 
Egg-white,  49 
Egg  albumin,  49 

crystallisation  of,  50 
Ehrlich's  test 

for  indol,  227 
Electrolytes,  action  of, 

on  colloids,  13 

on  heat  coagulation,  43 

on  ptyalin,   187 
Emulsification,  156,   157 
Emulsins,  184 
Emulsoids,  i 
Enantiomorphs,  150 


INDEX. 


395 


Enterokinase,  210 
Enzymes,  183 

amylopsin,  220 

autolytic,  228 

detection  of,  367 

erepsin,  218 

lactase,  221 

lipase,   158 

maltase,  221 

optimum  PH   for,  32,  184 

oxidase,  230 

pepsin,  20 1 

ptyalin,  186 

rennin,  207 

reversible  action  of,  185 

sucrase,  221 

trypsin,  210 

tyrosinase,  232 

urease,  287 
Erepsin,  218 
Erythrodextrin,  118 
Esbach's  albuminometer,    358 

reagent,  36 
Ethereal  sulphates,  281 

detection  of,  285 

estimation  of,  356,  358 

preparation  of,  285 
Euglobulin,  46 


Fats,   153 

digestion  of,  157 

emulsification  of,   156 

estimation  of,   169 

in  cheese,   172 

in  milk,   169 

saponification  of,   155 
Fatty  acid,   160 
Fehling's  method 

for  estimation  of  glucose,  171 
Fehling's  solution, 

preparation,    106 
Fehling's  test,  106 
Fermentation  test,   311 
Ferments, 

see  enzymes 
Fibrin,  ferment,  236 
Fibrinogen,  234 
F]our,   173 

Fluoride  plasma,  237 
Folin  and  Denis 

on  non-protein  nitrogen,   263 

on  sugar  in  milk,  172 
Folin  and  McEllroy 

method  for  sugar,  129 
Folin's  fume  absorber,  383 


Folin's  method 

for  ammonia,  330 

for  creatine,  338 

for  creatinine,  337 

for  cystine,  82 

for  sulphates,  355 
Folin-Schaffer  method 

for  uric  acid,  341 
Folin's  test 

for  uric  acid,  296 
Formol  titrations,  214 
Fraunhofer's  lines,  243 
Free  hydrochloric  acid,   196,^200 
Freezing  points,  5,  7 
Fructose,  101,  in 
Fruit  sugar,  see  fructose 
Fuld's  method 

for  pepsin,  204 
Fume-absorber,  383 
Furfurol,  112 
Fusion  mixture,  64 

Galactolipin,   165 
Galactose,  101,  165 
Gastric  juice,  194 

acidity  of,   198 

active  hydrochloric  acid,  196 

analysis  of,   197 

free  hydrochloric  acid,   196 

in  disease,   198 

mineral  chlorides  of,  200 

total  chlorides  of,  199 
Gelatin,  58,  365 
Gerhardt's  test,  316 
Gliadins,  34,   173 
Globulins, 

detection  of,  363 

eu-,  46 

in  muscle,  176 

in  serum,  46,  47 

preparation  of,  46,  47 

properties  of,  45 

pseudo-,  46 
Gluconic  acid,   104 
Glucoproteins,  57 
Glucosazone,    no 
Glucose,   101,   103 

estimation  of,  125,  251,  344 

fermentation  of,   311 

in  blood,  249 

in  urine,  308,  344 

phenyl-osazone  of,  no 

preparation  of,  105 

properties  of,  104,  in 
Glucosides,   103,   184 
Glutaminic  acid,  78 
Glutelins,  34,  173 


396 


INDEX. 


Gluten,  173 

Glycerol,  154,  159 

Glycerophosphoric  acid,  164 

Glycine,  72 

Glycine  ester  hydrochloride,  76 

Glycocohlic  acid,  264 

Glycogen,  123,  182 

estimation  of,  123 

identification  of,  366 

preparation,  124 

reactions  of,  124 
Glycoproteins,  34 
Giycosuria,  250,  309 
Glycuresis,  309 
Glycuronic  acid,  104 
Glyoxylic 

reaction,  39,  95 

reagent,  39 

Gmelin's  reaction,  268 
Graham  and  Poulton 

on  estimation  of  creatinine,  337 
Graham  on  gastric  contents,  198 
Grape-sugar,  see  glucose 
Guamne,  62,  65 
Guiaconic  acid,  232 
Guiacum  tincture, 

preparation  of,  232 
Gunsberg's  test,  197 


Haematin,  247 
Haematoporphyrin,  248 

in  urine,  279 
Haematuna,  305 
Haemin,  249 
Haemochromogen,  248 
Haemoglobin,  240 

in  urine,  305 
Haemolysis,  238 
Haser's  coefficient,  272 
Hay's  test  for  bile  salts,  266,  308 
Heat  coagulation,  42 
Heating  power,  standard,  136 
Heller's  test 

for  albumin,  49,  303 

for  blood,  306 
Hepatic  disease, 

ammonia  in,  301 

creatinine  in,  298 

urea  in,  286 

urobilin  in,  278,  280 
Hetero-albumose,  53 
Hetero-xanthine,  298 
Hexosans,   117 
Hexoses,  101 
Hippuric  acid,  77 
Histamine,  223 


Histidine, 

action  of  bacteria  on,  223 

preparation  of,  84 

properties  of,  86 
Histones,  34 
Hopkins-Cole 

method  for  uric  acid,  341 

test  for  proteins,  39 
Hopkins' 

method  for  uric  acid,  341 

test  for  lactic  acid,  180 
Huppert-Cole  test 

for  bile  pigments,  267 
Hurtley's  test 

for  aceto-acetic  acid,  317 
Hydrochloric  acid, 

active,  196 

in  gastric  juice,  195 

normal,  380 

specific  gravity  of,  377 
Hydrogen-ions, 

concentration  of,  14 

estimation  of,  29 
Hydrolysis 

of  fats,  155,  158,  161 

of  proteins,  70,  72,  84,  98 

of  starch,   118,  121 
Hypobromite, 

action  on  urea,  288,  362 

preparation  of,  290 
Hypoxanthine,  62,  65,  180 

in  urine,  298 

Indican,  318 
Indicators,  19 

preparation  of,  24 

ranges  of,  22 
Indol,  225 

aldehyde,  94 

from  tryptophane,  227 

mother  substance  of,  227 

tests  for,  227 
Indoxyl,  318 

Internal  compensation,  152 
Intestinal  extracts,  218 
Inulin,   in 
Invertase,  see  sucrase 
Invert  sugar,  in,  116 
Iodine  value,  156 
Iron 

in  haemoglobin,  241 

in  urine,  280 
Iso-electric  point, 

definition  of,   n,  31 

determination  of ,  1 1 

of  various  substances,  33 
Isoleucine,  98 


INDEX. 


397 


Jaffa's  test 

for  creatinine,   179,  300 

for  indican,  318 
Jones,  W.,  on  nucleic  acid,  64 

Kataphoresis,  9 
Kephalin,  165 
Kerasin,  165 
Keratin,  59 
Ketoses,  100 
Kjeldahl's  method,  321 
Kober's  colorimeter,  385 

Lactalbumin,  170 
Lactase,  221 
Lactic  acid,  180 

Hopkin's  test  for,  180 

in  gastric  contents,  195 

in  muscle,   181 

Uffelmann's  test  for,  181 
Lactosazone,  115,  171 
Lactose,  115,  171 

Cole's  test  for,  313 

distinction  from  glucose,  115,  171 

estimation  of,  139,  172 

in  milk,  171 

in   urine,    313 

osazone,   115,   171 
Laevulose,  101,  in 

see  also  fructose 
Laking  of  blood,  238 
Larrson's  method 

for  chlorides,  352 
Lecithin,    163 
Leucine,  97 

preparation  of,  98 

properties  of,  99 
Liebermann-Burchard  test 

for  cholesterol,  162 
Ling's  method  for  sugar,  141 
Ling's  indicator,   141 
Lipase,  158 
Lipines,  153 

Logarithms,  see  back  cover 
Long's  coefficient,  272 
Lysine,  69 

Maltase,  113,  184,  221 
in  pancreatic  extracts,  221 
intestinal,   221 

Malto-dextrin,  119 

Maltosazone,  115 

Maltose,  113 

action  of  enzymes  on,  221 
distinction  from  glucose,  114 
estimation  of,  129,  131,  134 
preparation  of,  113 


Mannite,   104 

Mannose,   101 

Meat,  see  muscle,  175 

Meig's  method  for  fat,  169 

Mellanby,  E., 

on  origin  of  creatinine,  298 
Mellanby,  J., 

on  amylopsin,  220 

on  estimation  of  trypsin,  214 

on  pancreatic  extracts,  212 

on  trypsin,  211 
Mercuric  nitrate, 

action  on  urea,  290 

action  on  proteins,  35 

preparation  of,  391 
Mercuric  sulphate  reagent, 

for  acetone  bodies,  347 

for  tryptophane,  84 
Mercury  green, 

rotations  with,  147 
Mesotartaric  acid,    152 
Metaproteins,  51 

detection  of,  363 
Methaemoglobin,  246 
Mett's  tubes,  205 

method  for  pepsin,  202,  205 
Micro-balance,  388 
Micro-methods 

for  chlorides  in  blood,  257 

for  sugar  in  blood,  253 

for  total  nitrogen,  329 
Milk,  1 66 

calcified,   206 

clotting,  167,  207 

composition  of,  167 

estimation  of  fat  in,  169 

estimation  of  lactose  in,  172 

inorganic  constituents  of,   172 
Milk  sugar,  see  lactose 
Millon's 

reaction,  38,  97 

reagent,  38 
Mineral  chlorides 

of  gastric  juice,  196,  200 
Molecular  weight 

of  casein,  166 

of  egg-albumin,  7 

of  haemaglobin,  241 
Molisch's  test,  41 
Monosaccharides,  100 
Morner's  reagent  for  tyrosine,  97 
Mucic  acid,  104 

test  for  lactose,  314 
Mucin,  57 
in  bile,  268 
in  saliva,   186 
preparation  of,  57 


398 


INDEX. 


Mucoid,  50 
Mulder's  test,  109 
Murexide  test,  295 
Muscle,  175 

extract,  176 
Mutarotation,  102 
Myosin,   176 

preparation  of,  177 
Myosinogen,  176 

Nephelometer,  385 
Neumann's   method 

for  organic  phosphorus,  169 
Neutral  salts,  action  of, 

on  colloids,  13 

on  heat  coagulation,  43 

on  ptyalin,   187 
Neutral  sulphur,  282 

estimation  of,  356,  358 
Ni col's  prism,  144 
Nitric  acid, 

action  on  proteins,  38,  49,  55 
Nitrogen, 

distribution  of,  270 

estimation  of,   321 

non-protein,  in  blood,  260 
Non-protein  nitrogen  of  blood, 

estimation  of,  260 
Normal  saline,  238 
Normal  solutions, 

preparation  of,  26,  380 
Nucleases,  61 
Nucleic  acid,  60 

hydrolysis  of,  65 

preparation  of,  64 
Nucleohistone,   60 
Nucleoproteins,  60 

detection  of,   364 

in  bile,  269 

preparation  of,  63 
Nucleosides,   61 
Nucleotides,  61 
Nylander's  test,  109 

Oleic  acid,  154 
Oliver's  test 

for  bile  salts,  266 
Optical  activity,  147 
Optically  active  compounds, 

resolution  of,   151 
Organic  phosphorus, 

detection  of,  169 
Osazone, 

of  glucose,  no 

of  lactose,  115 

of   maltose,    115 

preparation  of,  no 


Osmotic  pressure,  3-7 

oi  colloids,  7 

of  urine,  272 
Ost's  method  for  sugar, 

see  Wood-Ost 
Ovo-mucin,  49 
Ovo-mucoid,  50 
Oxalate  ot 

calcium,   172,  319 

urea,  288 

Oxalate  plasma,  237 
Oxidases,  230 
Oxy-butyric  acid,  314,  348 
Oxy-haemoglobin,  241 

crystallisation  of,  242 

in  urine,  305 

spectrum  of,  244 

Palmer 

on  acid  excretion,  275 
Palmitic  acid,  154 
Palmitin,   155 
Pancreas, 

extract  of,  88,  220 
Parabanic   acid,    292 
Paracasein,   167 
Paramyosinogen,  176 
Paraxanthine,  298 
Pavy's  method  for  sugar,  125 
Pentosans,  117 
Pentoses,  101 

in  urine,  312 
Pepsin,  201 

action  on  protein,  53,  202 

conditions  of  action  of,  201 

detection  of,  204 

distinction  from  rennin,  208 

estimation  of,   202 

law  of  action  of,  202 

optimum  reaction,  32,  201 

products  of  action,  53,  202 
Peptide  linkage,  41,  202 
Peptones,  52,  54,  56 

detection  of,  365 

formation  of,  202 

reactions  of,  56 

removal  from  fluids,  58 
Peroxidase,  231 
Peters,  Amos, 

method  for  sugar,  134 
Pettenkofer's  test,  265 
PH>  16 

PH,  determination  of,  30 
Phenol,  223,  282 
Phenol  red,  22 
Phenyl-alanine,  68 
Phenyl-osazone,  see  osazone 


INDEX. 


399 


Phosphates, 
acid,  283 

acid  potassium,  25 
calcium,  283 

distinction  from  proteins,  44 

earthy,  283 

estimation  of,  354 

in  milk,   172 

in  urine,  282,  319 

stellar,  319 

triple,  320 
Phosphatides,  162 
Phospholipins,    162 
Phosphoproteins,  34 
Phosphorus  in  proteins, 

detection  of,   169 
Phthalate, 

acid  potassium,  25 
Picramic  acid,  no 

preparation  of,  251 
Picric  acid, 

purification  of,  251,  336 
Pigments,  identification  of,  367 

of  bile,  267 

of  blood,  242-249 

of  muscle,   175 

of  urine,  278 
Piotrowski's  reaction,  40 
Pipettes, 

Dreyer's  dropping,  23 

method  of  discharging,  381 

Ostwald,  381 
Plasma, 

fluoride,  237 

oxalate,  237 

salted,  235 
Polarimetric    estimation    of   sugar, 

127 

Polarimeter,  description  of,   145 
Polarized  light,  143 
Polypeptides,  35 
Polysaccharides,   100,   117 
Potassium  permanganate, 

standardisation  of,  131 
Potatoes,   173 
Primary  albumoses,  53 
Prolamines,  34 
Protamines,  34 
Proteins,  33 

action  of  alcohol  on,  37 

bacterial  decomposition  of,  223 

carbohydrate  groups  of,  57    , 

classification  of,  33 

colour  reactions  of,  38 

conjugated,  34 

crystallisation  of,  50,  242 

definition,  33 


detection  of,  363 

general  reactions  of,  34 

heat  coagulation,  42,  44 

hydrolysed,  35 

hydrolysis  of,  70,  72,  84,  98,  202, 
211,  214,  218 

in  bile,   268 

in  urine,  302,  304 

of  muscle,   176 

osmotic  pressure  of,  7 

peptic  digestion  of,  53,  202 

percentage  composition  of,  33 

phosphorus  in,   169 

precipitants,  36,  37 

properties  of,  35-39 

removal  of,  48,  56,  250,  260 

sulphur  in,  41 

tryptic  digestion  of,  211 
Proteoses,  see  albumoses 
Prothrombin,   234 
Proto-albumose,  53 
Prout-Winter  method  for  chlorides, 

199 
Ptyalin,  action  of,  119,  187 

estimation  of,   188 
Purine  bases,  62 

in  meat,  179 

in  urine,  298 
Purpuric  acid,  295 
Putrescine,  225 
Pyrimidine  bases,  63 
Pyrollidone  carboxylic  acid,  80 

«* 

Racemic  compounds,  94,  151 
Raistrick  on   bacterial  decomposi- 
tion, 223 

Ranges  of  indicators,  22 
Reaction  of  fluids,  23 

of  urine,   273 

Reduced  alkaline  haematin,  248 
Reduced  oxalic  acid,  39 
Reducing  sugars,  106 
Removal  of  proteins,  10,  36,  37,  48, 

56 

Renal  disease,  263,  272,  275 
Rennet  ferment,  207 
Rennin,  207 

distinction  from  pepsin,  209 
Ribose,  101 
Robert's  test,  304 
Rotatory  power,  specific,  146 
Rothera's  test,  316 

Saccharic  acid,  104 
Saccharose,  see  cane  sugar 
Safranine  test,   109 
Saliva,  186 


400 


INDEX. 


Salivary  amylase,  183 
Salkowski's  test 

for  cholesterol,  162 
Salted  plasma,  236 
Salting  out,  161 
Saponification,   154 
Saponification  value,  154 
Sarcolactic  acid,   180 
Sarcosine,  178 
Scatol,  223,  225 
Scherer's  method,  358 
Schiff's  test,  296 
Schumm's  test,  306 
Schiitz-Borissow's  law,  202 
Scleroproteins,  34,  58 
Scott- Wilson  method,  349 
Secondary  albumoses,  54 
Sediments  in  urine,  319 
Seliwanoff's  test,  112 
Serum,  234 
Soaps,  154,  1 60,  161 
Sodium  hydroxide, 

standard   solutions   of,    26,    322, 

380 

Solids,  analysis  of,  370 
Soluble  myosin,   176 
Soluble  starch,  118,  391 
Sorbite,  104 

Sorensen  s  method,  70,  214 
Soya  bean  extract,  335 
Specific  gravity  of 

alcohol,  399 

anwnonia,  378 

hydrochloric  acid,  377 

milk,  167 

potassium  hydroxide,  378 

sodium  hydroxide,  378 

sulphuric  acid,   377 

urine,  271 

Specific  oxygen  capacity,  241 
Specific  rotatory  power,  146 
Spectroscope,  243 
Spiegler's  test,  304 
Standard  solutions  of 

buffers,  26 

hydrochloric  acid,  27,  380 

sodium  hydroxide,  26,  322,  380 
Starch,  117 

cellulose,  117 

digestion  of,  119,  187,  366 

grains,   119 

hydrolysis  of,  121 

reactions  of,    119,   174 

paste,  preparation  of,  120 

soluble,  preparation  of,  391 
Steapsin,  see  lipase 
Stearic  acid,  154 


Stearine,  160 
Stellar  phosphates,  319 
Stercobilin,  267 
Sterols,  154 
Stokes'  fluid,  153 
Sucrase,  116 
Sucrose,  221 
Sugars,  100 

estimation  of,  125 

in  blood,  250 

in  urine,  308,  312 

polarimetric  estimation  of,   127 

tolerance  for,  250 
Sulphates  in  urine,  281 

estimation  of,  355 
Sulphosalicylic  acid,  37 

test  for  albummuria,  304 
Sulphur 

estimation  of,  355 

in  proteins,  41 

in  urine,  281 

reaction  for  proteins,  41 
Sulphuric  acid, 

specific  gravity  of,  377 
Sulphur  test 

for  bile  salts,  266,  308 
Suspensoids,  i,  2 
Synalbumose,  53 


Tartaric  acid,  152 

Taurine,  265 

Teichmann's  crystals,  see  haemin 

Tensions  of  aqueous  vapour,  376 

Test  meal,  195 

Thio-albumose,  53 

Thrombin,  236 

Thrombokinase,  234 

Thymine,  63 

Tolerance  for  sugar,  250,  308 

Tollen's  test 

for  glycuronic  acid,  317 

for  pentoses,  312 
Torsion  balance,  388 
Total  nitrogen, 

estimation  of,  322 
Totani's  reaction,   87 
Triolein,  155 
Tripalmitin,  155 
Triple  phosphates,  320 
Tristearin,   155 

Trommer's  test  for  glucose,   105 
Trypsin,  210 

estimation  of,  214 

optimum  reaction  of, ^211 

preparation  of,  212 

products  of  action,  67,^211 


INDEX. 


401 


Tryptic  broth,  226 

Tryptophane,  40,  87 

action  of  bacteria  on,  226 
preparation,  87 
properties  of,  93,  217 

Tyrosinase,  232 

Tyrosine, 

action  of  bacteria  on,  223 
preparation  of,  95,  218 
properties,  96 

Uffelmann's  test 

for  lactic  acid,  181 
Uracil,  63 
Urates,  293 
Urea,  286 

constitution  of,  288 

detection  of,  290 

estimation  of,   334 

isolation  of,  291 

nitrate,  289 

oxalate,  288 
Uric  acid,  292 

crystals  of,  294,  297,  319 

estimation  of,  341 

in  urine,  297 

origin  of,  63,  294 
Urine, 

abnormal,  302 

acidity  of,  30,  273 

albumin  in,  302 

average  composition,  269 

deposits  in,  319 

diastase  in,  359 

inorganic  constituents,  280 

osmotic  pressure,  272 

PH,  30.   273 


pigments,  278 
proteins  in,  302 
reaction,  30,  273 
specific  gravity  of,  271 
sugar  in,  308 
total  nitrogen  of,  321 
total  solids  of,  272 

Urinometer,   272 

Urobilin,  267,  278 
tests  for,  279 

Urobilinogen,   267,   278 

Urochrome,  278 

Uroerythrin,  278 

Urorosein,   279 


Valine,  68 
Volhard's  method 
for  chlorides,  199, 


352 


Weights  and  measures,  375 
Werner 

on  urea,  288 
Weyl's  test 

for  creatinine,  179,  301 
Wheat  flour,  174 
Whey,  207 
Witte's  peptone,  52 
Wohlgemuth's   method 

for  diastase  in  urine,  359 

for  pytalin,  188,  192 
Wood-Ost's  method  for  sugar,  126 


Xanthine,  180 
Xanthoproteic  reaction,  38 
Xylose,  101 


CC 


'.   PRINTED  BY 

W.  HKFFER  AND  SONS  LTD. 
104,  Hiti.s  ROAD, 

CAMBRIDGE. 

; 


LOGARITHMS. 


Differences. 

01234 

5 

6 

7 

8 

9 

1 

234 

5  6 

7  8 

9 

10 

0000  0043  0086  0128  0170 

0212 

0253 

0294 

0334 

0374 

4 

8  12  17 

21  25 

29  33 

37 

11 

0414  0453  0492  0531  0569 

0607 

0645 

0682 

0719 

0755 

4 

8  11  15 

19  23 

26  30 

34 

12 

0792  0828  0864  0899  0934 

0969 

1004 

1038 

1072 

1106 

3 

7  10  14 

17  21 

24  28 

31 

13 

1139  1173  1206  1239  1271 

1303 

1335 

1367 

1399 

1430 

3 

6  10  13 

16  19 

23  26 

29 

14 

1461  1492  1523  1553  1584 

1614 

1644 

1673 

1703 

1732 

3 

6  9  12 

15  18 

21  24 

27 

15 

1761  1790  1818  1847  1875 

1903 

1931 

1959 

1987 

2014 

3 

6  8  11 

14  17 

20  22 

25 

16 

2041  2068  2095  2122  2148 

2175 

2201 

2227 

2253 

2279 

3 

5  8  11 

13  16 

18  21 

24 

17 

2304  2330  2355  2380  2405 

2430 

2455 

2480 

2504 

2529 

2 

5  7  10 

12  15 

17  20 

22 

18 

2553  2577  26C1  2625  2648 

2672 

2695 

2718 

2742 

2765 

2 

579 

12  14 

16  19 

2  1 

19 

2788  2810  2833  2856  2878 

2900 

2923 

2945 

2967 

2989 

9 

479 

11  13 

16  18 

20 

20 

3010  3032  3054  3075  3096 

3118 

3139 

3160 

3181 

3201 

2 

468 

11  13 

15  17 

19 

21 

3222  3243  3263  3284  3304 

3324 

3345 

3365 

3385 

3404 

2 

468 

10  12 

14  16 

18 

22 

3424  3444  3464  3483  3502 

3522 

3541 

3560 

3579 

3598 

2 

468 

10  12 

14  15 

17 

23 

3617  3636  3655  3674  3692 

3711 

3729 

3747 

3766 

3784 

2 

467 

9  11 

13  15 

17 

24 

3802  3820  3838  3856  3874 

3892 

3909 

3927 

3945 

3962 

2 

457 

9  11 

12  14 

16 

25 

3979  3997  4014  4031  4048 

4065 

4082 

4099 

4116 

4133 

2 

357 

9  10 

12  14 

15 

26 

4150  4166  4183  4200  4216 

4232 

4249 

4265 

4281 

4298 

2 

357 

8  10 

11  13 

15 

27 

4314  4330  4346  4362  4378 

4393 

4409 

4425 

4440 

4456 

2 

356 

8  9 

11  13 

14 

28 

4472  4487  4502  4518  4533 

4548 

4564 

4579 

4594 

4609 

2 

356 

8  9 

11  12 

14 

29 

4624  4639  4654  4669  4683 

4698 

4713 

4728 

4742 

4757 

1 

346 

7  9 

10  12 

13 

30 

4771  4786  4800  4814  4829 

4843 

4857 

4871 

4886 

4900 

1 

346 

7  9 

10  11 

13 

31 

4914  4928  4942  4955  4969 

4983 

4997 

5011 

5024 

5038 

1 

346 

7  8 

10  11 

12 

32 

5051  5065  5079  5092  5105 

5119 

5132 

5145 

5159 

5172 

1 

345 

7  8 

9  11 

12 

33 

5185  5198  5211  5224  5237 

5250 

5263 

5276 

5289 

5302 

1 

345 

6  8 

9  10 

12 

34 

5315  5328  5340  5353  5366 

5378 

5391 

5403 

5416 

5428 

1 

345 

6  8 

9  10 

11 

35 

5441  5453  5465  5478  5490 

5502 

5514 

5527 

5539 

5551 

1 

245 

6  7 

9  10 

11 

36 

5563  5575  5587  5599  5611 

5623 

5635 

5647 

5658 

5670 

1 

245 

6  7 

8  10 

11 

37 

5682  5694  5705  5717  5729 

5740 

5752 

5763 

5775 

5786 

I 

235 

6  7 

8  9 

10 

38 

5798  5809  5821  5832  5843 

5855 

5866 

5877 

5888 

5899 

1 

235 

6  7 

8  9 

10 

39 

5911  5922  5933  5944  5955 

5966 

5977 

5988 

5999 

6010 

1 

234 

5  7 

8  9 

10 

40 

6021  6031  6042  6053  6064 

6075 

6085 

6096 

6107 

6117 

I 

234 

5  6 

8  9 

10 

41 

6128  6138  6149  6160  6170 

6180 

6191 

6201 

6212 

6222 

1 

234 

5  6 

7  8 

9 

42 

6232  6243  6253  6263  6274 

6284 

6294 

6304 

6314 

6325 

1 

234 

5  6 

7  8 

9 

43 

6335  6345  6355  6365  6375 

6385 

6395 

6405 

6415 

6425 

1 

?  3  4 

5  6 

7  8 

9 

44 

6435  6444  6454  6464  6474 

6484 

6493 

6503 

6513 

6522 

1 

234 

5  6 

7  8 

9 

45 

6532  6542  6551  6561  6571 

6580 

6590 

6599 

6609 

6618 

1 

234 

5  6 

7  8 

9 

46 

6628  6637  6646  6656  6665 

6675 

6684 

6693 

6702 

6712 

1 

234 

5  6 

7  7 

8 

47 

6721  6730  6739  6749  6758 

6767 

6776 

6785 

6794 

6803 

1 

234 

5  5 

6  7 

8 

48 

6812  6821  6830  6839  6848 

6857 

6866 

6875 

6884 

6893 

1 

234 

4  5 

6  7 

8 

49 

6902  6911  6920  6928  6937 

6946 

6955 

6964 

6972 

6981 

1 

234 

4  5 

6  7 

8 

50 

6990  6998  7007  7016  7024 

7033 

7042 

7050 

7059 

7067. 

1 

233 

4  5 

6  7 

8 

51 

7076  7084  7093  7101  7110 

7118 

7126 

7135 

7143 

7152 

1 

233 

4  5 

6  7 

8 

52 

7160  7168  7177  7185  7193 

7202 

7210 

7218 

7226 

7235 

1 

223 

4  5 

6  7 

7 

53 

7243  7251  7259  7267  7275 

7284 

7292 

7300 

7308 

7316 

1 

223 

4  5 

6  6 

7 

54 

7324  7332  7340  7348  7356 

7364 

7372 

7380 

7388 

7396 

1 

223 

4  5 

6  6 

7 

01234 

5 

6 

7 

8 

9 

1 

234 

5  6 

7  8 

9 

LOGARITHMS. 


Differences. 

01234    5678 

9 

1 

2 

3 

4 

5 

6 

7 

8 

9 

55 

7404  7412  7419  7427  7435  7443  7451  7459  7466 

7474 

1 

2 

2 

3 

4 

5 

5 

6 

7 

56 

7482  7490  7497  7505  7513  7520  752S  7536  7543 

7551 

1 

2 

2 

3 

4 

5 

5 

6 

7 

57 

7559  7566  7574  7582  75S9  7597  7604  7612  7619 

7627 

1 

2 

2 

3 

4 

5 

5 

6 

7 

58 

7634  7642  7649  7657  7664  7672  7679  7686  7694 

7701 

1 

1 

2 

3 

4 

4 

5 

6 

7 

59 

7709  7716  7723  7731  7738  7745  7752  7760  7767 

7774 

1 

1 

2 

3 

4 

4 

5 

6 

7 

60 

7782  7789  7796  7803  7810  7818  7825  7832  7839 

7846 

1 

1 

2 

3 

4 

4 

5 

6 

6 

61 

7853  7860  7868  7875  7882  7889  7896  7903  7910 

7917 

1 

1 

2 

3 

4 

4 

5 

6 

6 

62 

7924  7931  7938  7945  7952  7959  7966  7973  7980 

7987 

1 

1 

2 

3 

3 

4 

5 

6 

6 

63 

7993  8000  8007  8014  8021  8028  8035  8041  8048 

8055 

1 

1 

2 

3 

3 

4 

5 

5 

6 

64 

8062  8069  8075  8082  8089  8096  8102  8109  8116 

8122 

1 

1 

2 

3 

3 

4 

5 

5 

6 

65 

8129  8136  8142  8149  8156  8162  8169  8176  8182 

8189 

1 

1 

2 

3 

3 

4 

5 

5 

6 

66 

8195  8202  8209  8215  8222  8228  8235  8241  8248 

8254 

1 

1 

2 

3 

3 

4 

5 

5 

6 

67 

8261  8267  8274  8280  8287  8293  8299  8306  8312 

8319 

1 

1 

2 

3 

3 

4 

5 

5 

6 

68 

8325  8331  8338  8344  8351  8357  8363  8370  8376 

8382 

1 

1 

2 

3 

3 

4 

4 

5 

6 

69 

8388  8395  8401  8407  8414  8420  8426  8432  8439 

8445 

1 

1 

2 

2 

3 

4 

4 

5 

6 

70 

8451  8457  8463  8470  8476  8482  8488  8494  8500 

8506 

1 

1 

2 

2 

3 

4 

4 

5 

6 

71 

8513  8519  8525  8*531  8537  8543  8549  8555  8561 

8567 

1 

1 

2 

2 

3 

4 

4 

5 

5 

72 

8573  8579  8585  8591  8597  8603  8609  8615  8621 

8627 

1 

1 

2 

2 

3 

4 

4 

5 

5 

73 

8633  8639  8645  8651  8657  8663  8669  8675  8681 

8686 

1 

1 

2 

2 

3 

4 

4 

5 

5 

74 

8692  8698  8704  8710  8716  8722  8727  8733  8739 

8745 

1 

1 

2 

2 

3 

4 

4 

5 

5 

75 

8751  8756  8762  8768  8774  8779  8785  8791  8797 

8802 

I 

1 

2 

2 

3 

3 

4 

5 

5 

76 

8808  8814  8820  8825  8831  8837  8842  8848  8854 

8859 

1 

1 

2 

2 

3 

3 

4 

5 

5 

77 

8865  8871  8876  8882  8887  8893  8899  8904  8910 

8915 

1 

1 

2 

2 

3 

3 

4 

4 

5 

78 

8921  8927  8932  8938  8943  8949  8954  8960  8965 

8971 

1 

1 

2 

2 

3 

3 

4 

4 

5 

79 

8976  8982  8987  8993  8998  9004  9009  9015  9020 

9025 

1 

1 

2 

2 

3 

3 

4 

4 

5 

80 

9031  9036  9042  9047  9053  9058  9063  9069  9074 

9079 

1 

1 

2 

2 

3 

3 

4 

4 

5 

81 

9085  9090  9096  9101  9106  9112  9117  9122  9128 

9133 

1 

1 

2 

2 

3 

3 

4 

4 

5 

82 

9138  9143  9149  9154  9159  9165  9170  9175  9180 

9186 

1 

1 

2 

2 

3 

3 

4 

4 

5 

83 

9191  9196  9201  9206  9212  9217  9222  9227  9232 

9238 

1 

1 

2 

2 

3 

3 

4 

4 

5 

84 

9243  9248  9253  9258  9263  9269  9274  9279  9284 

9289 

1 

1 

2 

2 

3 

3 

4 

4 

5 

85 

9294  9299  9304  9309  9315  9320  9325  9330  9335 

9340 

1 

1 

2 

2 

3 

3 

4 

4 

5 

86 

9345  9350  9355  9360  9365  9370  9375  9380  9385 

9390 

1 

1 

1 

2 

3 

3 

4 

4 

5 

87 

9395  9400  9405  9410  9415  9420  9425  9430  9435 

9440 

0 

1 

1 

2 

2 

3 

3 

4 

4 

88 

9445  9450  9455  9460  9465  9469  9474  9479  9484 

9489 

0 

1 

1 

2 

2 

3 

3 

4 

4 

89 

9494  9499  950^  9509  9513  9518  9523  9528  9533 

9538 

0 

1 

1 

2 

2 

3 

3 

4 

4 

93 

9542  9547  9552  9557  9562  9566  9571  9576  9581 

9586 

0 

1 

1 

2 

2 

3 

3 

4 

4 

91 

9590  9595  9600  9605  9609  9614  9619  9624  9628 

9633 

0 

1 

1 

2 

2 

3 

3 

4 

4 

92 

9638  9643  9647  9652  9657  9661  9666  9671  9675 

9680 

0 

1 

1 

2 

2 

3 

3 

4 

4 

93 

9685  9689  9694  9699  9703  9708  9713  9717  9722 

9727 

0 

1 

1 

2 

2 

3 

3 

4 

4 

94 

9731  9736  9741  9745  9750  9754  9759  9763  9768 

9773 

0 

1 

1 

2 

2 

3 

3 

4 

4 

95 

9777  9782  9786  9791  9795  9800  9805  9809  9814 

9818 

0 

1 

2 

2 

3 

3 

4 

4 

96 

9823  9827  9832  9836  9841  9845  9850  9854  9859 

9863 

0 

1 

2 

2 

3 

3 

4 

4 

97 

9868  9872  9877  9881  9886  9890  9894  9899  9903 

9908 

0 

1 

2 

2 

3 

3 

4 

4 

98 

9912  9917  9921  9926  9930  9934  9939  9943  9948 

9952 

0 

1 

2 

2 

3 

3 

4 

4 

99 

9956  9961  9965  9969  9974  9978  9983  9987  9991 

9996 

0 

1 

2 

2 

3 

3 

3 

4 

01234    5678 

9 

1 

2 

3 

4 

5 

6 

7 

8 

9 

X3 


Cole,   S/  .  42254 

tactical  physiological 
chemistry.        5th  ed.... 


